Compositions and methods to treat solid tumors

ABSTRACT

Compositions and methods that utilize anti-CD11b antibodies, anti-CD18 antibodies, anti-myeloperoxidase (MPO) antibodies, anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximab, neutrophil inhibitory factor (NIF) protein, and/or combinations thereof to treat solid tumors are described.

CROSS REFERENCE TO RELATED APPLICATION

This is a division of co-pending U.S. application Ser. No. 15/338,092,filed Oct. 28, 2016, which claims the benefit of the earlier filing dateof U.S. Provisional Patent Application No. 62/248,144 filed on Oct. 29,2015, and of U.S. Provisional Patent Application No. 62/332,397 filed onMay 5, 2016, each of which is incorporated herein by reference in theirentirety as if fully set forth herein.

FIELD OF THE DISCLOSURE

The present disclosure provides compositions and methods to treat solidtumors. The compositions and methods utilize anti-CD11b antibodies,anti-CD18 antibodies, anti-myeloperoxidase (MPO) antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximab,neutrophil inhibitory factor (NIF) protein, and/or combinations thereof,as well as other compounds disclosed herein.

BACKGROUND OF THE DISCLOSURE

A “tumor” is a swelling or lesion formed by the growth and division oftumor cells. “Tumor cells” are abnormal cells that divide in anuncontrolled manner and generally continue to divide after the stimulithat initiated growth and division ceases.

Tumors usually show partial or complete lack of structural organizationand functional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be either benign, pre-malignant, ormalignant. For example, fibromas, also known as fibroid tumors orfibroids, are composed of fibrous or connective tissue and are usuallybenign tumors. Despite being benign, such tumors often requiretherapeutic intervention.

Cancer (medical term: malignant neoplasm) refers to a class of diseasesin which a group of cells display uncontrolled growth (division beyondthe normal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis. “Metastasis” refers to the spread ofcancer cells from their original site of proliferation to another partof the body. For solid tumors, the formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood or lymph, infiltration oftarget organs. Finally, the growth of a new tumor, i.e. a secondarytumor or metastatic tumor, at the target site depends on angiogenesis.Tumor metastasis often occurs even after the removal of the primarytumor because tumor cells (including cancer stem cells) or componentsmay remain and develop metastatic potential.

Cancerous solid tumors can be found, for example, in the bone, bladder,brain, breast, colon, esophagus, gastrointestinal tract, genito-urinarytract, kidney, liver, lung, nervous system, ovary, pancreas, prostate,retina, skin, stomach, testicles, and/or uterus.

Taking ovarian cancer as an example, this type of cancer is currentlyregarded as one of the most common cancer types among women. In 2012alone, the number of new cases of ovarian cancer stood at 239,000,constituting 1.7% of all cancer cases worldwide, according to WorldCancer Research Fund International.

Ovarian cancer is rarely diagnosed at its early stages, making thetreatment of this cancer at an advanced stage difficult. Chemotherapyfor ovarian cancer is most often a combination of 2 or more drugs, givenIV every 3- to 4-weeks. Giving combinations of drugs rather than justone drug alone seems to be more effective in the initial treatment ofovarian cancer. The standard approach is the combination of a platinumcompound, such as cisplatin or carboplatin, and a taxane, such aspaclitaxel (Taxol®, Bristol-Myers Squibb, New York N.Y.) or docetaxel(commercially available as Taxotere®, Aventis Pharma, Antony, France).However, the use of chemotherapeutic agents across all cancer typessuffers from two major limitations. First, chemotherapeutic agents arenot specific for cancer cells and particularly at high doses, they aretoxic to normal rapidly dividing cells. Second, with time and repeateduse, cancer cells develop resistance to chemotherapeutic agents therebyproviding no further benefit to the patient. Furthermore, as thesurvival rate among women suffering from ovarian cancer is poor, effortsare afoot to develop new medications and therapies for the effectivetreatment of this disease.

SUMMARY OF THE DISCLOSURE

The present disclosure advances the treatment of solid tumors, includingovarian cancer solid tumors, by utilizing anti-CD11b antibodies,anti-CD18 antibodies, anti-myeloperoxidase (MPO) antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximaband/or neutrophil inhibitory factor (NIF) protein. Other compoundsdisclosed herein may also be used.

Use of the described compounds has been shown to have anti-cancereffects in numerous cancer models, including in chemosensitive cancercells, chemoresistant cancer cells, and cancer stem cells. Thus use ofthe compounds are broadly effective against varied cancer cell types. Inaddition, the compounds can be used in combination with each other andin combination with chemotherapeutic agents to provide effectivetherapeutic treatments. Use in combination with chemotherapeutic agentsmay allow lowering the dose of chemotherapeutic agents.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E. Anti-cancer effects of Abciximab and NIF. (FIG. 1A)Anti-cancer effects of Abciximab in sensitive EOC cell lines (A2780,MDAH-2774, SKOV-3, OV90, TOV21G, OV112D, OV433, and HTB-161) and theirTaxotere or Cisplatin resistant counterparts, human macrophages, andother cancer cell lines including colon (HTB-37), endometrial(CRL-1671), lung (CCL-257), prostate (CRL-1740), bladder (HTB-4), andhepatocellular (HB-8065) cancers, in vitro. (FIG. 1B) The combinedeffect of Abciximab alone or in combination with Cisplatin in bothsensitive A2780 and their (FIG. 1C) Cisplatin resistant counterpart invitro. (FIG. 1D) The synergistic effect of Abciximab and Cisplatin inboth sensitive A2780 and their Cisplatin resistant counterpart in vitro,with Cl<1 indicating synergism of the combined drugs. (FIG. 1E)Anti-cancer effects of NIF in both sensitive A2780 and their Cisplatinresistant counterpart in vitro.

FIGS. 2A and 2B. Real-time RT-PCR and Western Blot analysis of integrinαV and integrin β1 levels in (FIG. 2A) A2780 sensitive and (FIG. 2B)Cisplatin resistant EOC cells.

FIGS. 3A-3E. Cell viability in A2780 sensitive and cisplatin resistantEOC cells after treatment with integrin αV and integrin β1 antibody.(FIG. 3A) A2780 chemosensitive and (FIG. 3B) cisplatin resistant EOCcells were treated with or without integrin αV and integrin β1 antibodyalone or in combination and viability was assessed using the TACS MTTCell Proliferation Assay. (FIG. 3C) A2780 chemosensitive and (FIG. 3D)cisplatin resistant EOC cells were treated with or without a combinationof integrin αV and integrin β1 antibody with or without Cisplatin andviability was assessed using the TACS MTT Cell Proliferation Assay whilesynergism (FIG. 3E) was assessed with the Compusyn software with Cl<1indicating a synergistic effect of the antibodies and Cisplatin.

FIGS. 4A and 4B. Integrin αV and integrin β1 antibodies cross-react witha potential soluble target in EOC cell media. To test whether mediacollected from A2780 EOC cells contains a target that cross-reacts withthe integrin αV or integrin β1 monoclonal antibodies, cells werecultured for 48 hours followed by removal of media. Either mouseanti-integrin αV or mouse anti-integrin β1 antibody was incubated withthe conditioned cell culture media for 24 hours to block the antibodywith the unknown target. The blocked antibody media was used to treatA2780 sensitive (FIG. 4A) or cisplatin resistant (FIG. 4B) EOC cells foranother 24 hours followed by assessment of viability by the TACS MTTCell Proliferation Assay. No decrease in cell viability was observedwhen treating with media containing “blocked” antibody in cisplatinresistant cells and in sensitive cells, but to a much lesser extent.

FIG. 5. Real-time RT-PCR analysis of MPO in multiple cancer cell lines.MPO mRNA level was determined in epithelial ovarian cancer (EOC)(SKOV-3, MDAH-2774, OVCAR-3, OV-21, OV-90), pancreatic cancer (BXPC-3),colon cancer (COLO-3), non-Hodgkin's B cell lymphoma (DLCL-2), bladdercancer (HTB-4), and endometrial cancer (CRL-1671) cell lines.

FIG. 6. Immunofluorescence detection of CD11b or MPO in normal ovariantissue and ovarian cancer tissues and cells. Normal ovarian tissues,cancerous ovarian tissues, and ovarian cancer cells (MDAH-2774 andSKOV-3) were immunofluorescently stained for either CD11b or MPO.

FIGS. 7A and 7B. Real-time RT-PCR analysis of CD11b and MPO in cells.(FIG. 7A) CD11b and (FIG. 7B) MPO mRNA levels were determined in bothHOSEpiC and EOC cell lines.

FIG. 8. CD11b expression in normal and cancerous tissues. Tissuesections were incubated with the FITC-conjugated antibody (mouseanti-CD11b monoclonal antibody; Santa Cruz Biotechnology) diluted at1:100 ratio for 1 hour at room temperature followed by visualizationwith the Axiovert 25 inverted microscope (Zeiss).

FIG. 9. CD11b and MPO expression in cancer stem-like cells isolated fromthe EOC cell line, SKOV-3. Isolation of CSCs was achieved utilizing themagnetic-activated cell sorting technique for CD44+CD117+ cells via aCD44 or CD117 antibody coupled to magnetic beads. Lysates from cells wasthen immunoprecipitated for either CD11b or MPO, and protein wasseparated by SDS PAGE/Western blot and transferred to PVDF membrane.Protein was probed with either CD11b or MPO antibody. Bands wereanalyzed with Image J and are relative to SKOV-3 bands.

FIGS. 10A and 10B. CD11b antibody increases apoptosis in EOC cells. TwoEOC cell lines, MDAH-2774 and SKOV-3, were treated for 24 hours withCD11b antibody (15 μg/ml). (FIG. 10A) There was a significant increasein caspase-3 activity in both EOC cell lines, suggesting an increase inapoptosis. (FIG. 10B) There was an increase in the degree offluorescence in CD11b antibody treated EOC cells, which indicates anincrease in fragmented DNA and apoptosis.

FIG. 11. MPO antibody binds to CD11b in ovarian cancer cells. Celllysates from either MDAH-2774 or SKOV-3 EOC cells wereimmunoprecipitated with MPO antibody. The proteins were separated by SDSPAGE/Western blot and transferred to a PVDF membrane. The membrane wasprobed with anti-CD11b antibody.

FIG. 12. Scratch assay to determine inhibition of migration of EOCcells. Phase-contrast optical images of the SKOV-3 EOC cell line at 0,6, and 12, 18, and 24 hours after the creation of a cell-free zone usingthe scratch technique. At time 0, there was a 1 mm wide cell free zoneobtained utilizing a pipette tip to scratch away adherent SKOV-3 EOCcells. Treatment with CD11b antibody resulted in a significantly slowerrate of migration of the EOC cells and eventual cell death around 24hours.

FIG. 13. Treatment with CD11b antibody prevented an increase in tumorvolume in athymic NUDE mice. Control (n=2) and treated mice (n=2)obtained from Harlan were subcutaneously injected on each flank withsaline or a single-cell suspension of the human ovarian cancer cell lineSKOV-3 using a 21 gauge needle. Once the tumor was visible, mice wereinjected daily with 0.12 ml of CD11b antibody into the tumor and tumormeasurement were taken daily for 5 days. Tumor weights were calculatedas Tumor weight (mg)=(A×B2)/2, where A and B are the tumor length andwidth (in mm), respectively.

FIGS. 14A-14E. Real-time RT-PCR and Immunoprecipitation/Western Blotanalysis of CD11b, CD18 and MPO in sensitive as compared tochemoresistant EOC cells. Chemoresistant EOC cell lines (0.3 μM Taxotere(Tx) or 1.5 μM cisplatin (cis)) and their chemosensitive (sens)counterparts were utilized to determine (FIG. 14A) MPO mRNA and (FIG.14B) protein levels. Chemoresistant EOC cell lines and theirchemosensitive counterparts were also utilized to determine CD11b (FIG.14C) mRNA and (FIG. 14D) protein levels, as well as (FIG. 14E) CD18 mRNAlevels.

FIGS. 15A-15D. Anti-cancer effects of the CD11b antibody. (FIG. 15A)Anti-cancer effects of CD11b antibody in sensitive EOC cell lines(A2780, MDAH-2774, SKOV-3, OV90, TOV21G, OV112D, OV433, and HTB-161) andtheir Taxotere or Cisplatin resistant counterparts, non-cancer cells(human macrophages (EL1) and HOSEpiC), and other cancer cell linesincluding colon (HTB-37), endometrial (CRL-1671), lung (CCL-257),prostate (CRL-1740), bladder (HTB-4), and hepatocellular (HB-8065), invitro. (FIG. 15B) The combined effect of CD11b antibody in combinationwith Cisplatin in both sensitive A2780 and their (FIG. 15C) Cisplatinresistant counterpart in vitro. (FIG. 15D) The synergistic effect ofCD11b antibody and Cisplatin in both sensitive A2780 and their Cisplatinresistant counterpart in vitro, with Cl<1 indicating synergism of thecombined drugs. (FIG. 1F) Anti-cancer effects of NIF in both sensitiveA2780 and their Cisplatin resistant counterpart in vitro.

FIG. 16. CD11b antibody cross-reacts with a potential soluble target inEOC cell media. To test whether media collected from A2780 EOC cellscontains a target that cross-reacts with the CD11b monoclonal antibody,cells were cultured for 48 hours followed by removal of media. The CD11bantibody was incubated with the conditioned cell culture media for 24hours to block the antibody with the unknown target. The blockedantibody media was used to treat A2780 sensitive EOC cells for another24 hours followed by assessment of viability by the TACS MTT CellProliferation Assay. No decrease in cell viability was observed whentreating with media from cells containing “blocked” antibody.

FIGS. 17A-17D. Cell viability in sensitive and cisplatin resistant EOCcells after treatment with MPO antibody. (FIG. 17A) Sensitive andcisplatin resistant MDAH-2774 and SKOV-3 as well as cancer stem cellsderived from SKOV-3 were treated with increasing doses of MPO antibodyand viability was assessed by the TACS MTT Cell Proliferation Assay.(FIG. 17B) Sensitive and cisplatin resistant A2780 EOC cells as well ashuman macrophage cells were treated with MPO antibody and viability wasassessed by the TACS MTT Cell Proliferation Assay. (FIG. 17C) SKOV-3sensitive and (FIG. 17D) SKOV-3 Cisplatin resistant EOC cells weretreated with a combination of MPO antibody and cisplatin and viabilitywas assessed with the

FIG. 18. Real-time RT-PCR analysis of TLR4 in sensitive andchemoresistant EOC cells. The commercial cisplatin chemoresistant A2780EOC cell line (1.0 μM), as well as derived Taxotere® (0.3 μM) orcisplatin (1.5 μM) resistant MDAH-2774 and SKOV-3 EOC cell lines andtheir chemosensitive counterparts were utilized to determinechemoresistant as compared to their chemosensitive counterparts.

FIG. 19. Exemplary CD11b sequence (GenBank: AH004143.2; SEQ ID NO: 1).

FIG. 20. Exemplary CD18 sequence (GenBank: ADS87820.1; SEQ ID NO: 2).

FIGS. 21A-21D. Exemplary Abciximab sequences including its heavy chain 1(FIG. 25A; SEQ ID NO: 3); light chain 1 (FIG. 25B; SEQ ID NO: 4); heavychain 2 (FIG. 25C; SEQ ID NO: 5); and light chain 2 (FIG. 25D; SEQ IDNO: 6).

FIG. 22. Exemplary nucleotide sequence encoding Neutrophil inhibitoryfactor (NIF; SEQ ID NO: 7).

FIG. 23. Table 1. Oligonucleotide primers (SEQ ID NOs: 9-18) describedin Example 2.

REFERENCE TO SEQUENCE LISTING

The nucleic acid sequences described herein are shown using standardletter abbreviations for nucleotide bases, as defined in 37 C.F.R. §1.822. A computer readable text file, entitled “Sequence Listing.txt”created on or about May 18, 2019, with a file size of 40 KB, containsthe sequence listing for this application and is hereby incorporated byreference in its entirety.

DETAILED DESCRIPTION

A “tumor” is a swelling or lesion formed by the growth and division oftumor cells. “Tumor cells” are abnormal cells that divide in anuncontrolled manner and generally continue to divide after the stimulithat initiated growth and division ceases.

Tumors usually show partial or complete lack of structural organizationand functional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be either benign, pre-malignant, ormalignant.

Fibrosis is the pathological state of excess deposition of fibroustissue, as well as the process of connective tissue deposition inhealing. Fibroid tissue solid tumors (e.g., fibromas, fibroids, fibroidtumors) are the result of formations of excess fibrous connective tissuein an organ or tissue in a reparative or reactive process. Fibroidtissue solid tumors are usually benign. Even though benign, these tumorscan cause a number of adverse side effects, such as pain and/or damageto the architecture and function of the underlying affected organ ortissue. Examples of conditions that can be associated with fibroidtissue solid tumors include some forms of adhesive capsulitis, arterialfibrosis, arthrofibrosis, Crohn's disease, cirrhosis, cystic fibrosis,endomyocardial fibrosis, fibrous cysts, idiopathic pulmonary fibrosis,keloids, mediastinal fibrosis, myelofibrosis, nephrogenic systemicfibrosis, Peyronie's disease, pulmonary fibrosis, progressive massivefibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis,uterine fibroids (uterine leiomyomas), and other precancerous fibromas.Fibrosarcoma is a malignant solid tumor derived from fibrous connectivetissues.

With respect to uterine fibroids particularly, The National UterineFibroids Foundation estimates that as many as 80% of all women in theUnited States have uterine fibroids, and one in four of these women havesymptoms severe enough to require treatment. The current standards oftreatment are hormone therapy, oral contraceptives, gonadotropinreleasing hormone agonists, uterine artery embolization and myomectomy.

Cancer (medical term: malignant neoplasm) refers to a class of diseasesin which a group of cells display uncontrolled growth (division beyondthe normal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis. “Metastasis” refers to the spread ofcancer cells from their original site of proliferation to another partof the body. For solid tumors, the formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood or lymph, infiltration oftarget organs. Finally, the growth of a new tumor, i.e. a secondarytumor or metastatic tumor, at the target site depends on angiogenesis.Tumor metastasis often occurs even after the removal of the primarytumor because tumor cells (including cancer stem cells) or componentsmay remain and develop metastatic potential.

Cancerous solid tumors can be found, for example, in the bone, bladder,brain, breast, colon, esophagus, gastrointestinal tract, genito-urinarytract, kidney, liver, lung, nervous system, ovary, pancreas, prostate,retina, skin, stomach, testicles, and/or uterus.

Taking ovarian cancer as an example, this type of cancer is currentlyregarded as one of the most common cancer types among women. In 2012alone, the number of new cases of ovarian cancer stood at 239,000,constituting 1.7% of all cancer cases worldwide, according to WorldCancer Research Fund International.

Ovarian cancer is rarely diagnosed at its early stages, making thetreatment of this cancer at an advanced stage difficult. Chemotherapyfor ovarian cancer is most often a combination of 2 or more drugs, givenIV every 3- to 4-weeks. Giving combinations of drugs rather than justone drug alone seems to be more effective in the initial treatment ofovarian cancer. The standard approach is the combination of a platinumcompound, such as cisplatin or carboplatin, and a taxane, such aspaclitaxel (Taxol®, Bristol-Myers Squibb, New York N.Y.) or docetaxel(commercially available as Taxotere®, Aventis Pharma, Antony, France).Use of chemotherapeutic agents across all cancer types, however, suffersfrom two major limitations. First, chemotherapeutic agents are notspecific for cancer cells and particularly at high doses, they are toxicto normal rapidly dividing cells. Second, with time and repeated use,cancer cells develop resistance to chemotherapeutic agents therebyproviding no further benefit to the patient.

As the survival rate among women suffering from ovarian cancer is poor,efforts are afoot to develop new medications and therapies for theeffective treatment of this disease.

The present disclosure advances the treatment of solid tumors, includingovarian cancer solid tumors, by utilizing anti-CD11b antibodies,anti-CD18 antibodies, anti-MPO antibodies, anti-integrin αV antibodies,anti-integrin β1 antibodies, Abciximab and/or neutrophil inhibitoryfactor (NIF) protein. Other compounds disclosed herein may also be used.

Use of the described compounds has been shown to have anti-cancereffects in numerous cancer models, including in chemosensitive cancercells, chemoresistant cancer cells, and cancer stem cells. Thus use ofthe compounds are broadly effective against varied cancer cell types. Inaddition, the compounds can be used in combination with each other andin combination with chemotherapeutic agents to provide effectivetherapeutic treatments.

CD11b (Mac-1α; integrin αM chain) is part of the CD11b/CD18 heterodimer(Mac-1α, Mβ2 integrin), also known as the C3 complement receptor. Itfunctions as a receptor for complement (C3bi), fibrinogen, or clottingfactor X. In humans, CD11b is strongly expressed on myeloid cells andweakly expressed on NK cells and some activated lymphocytes as well ason microglia in the brain. In mice, the CD11b antigen is expressed onmonocytes/macrophages and microglia. To a lower extent it is expressedon granulocytes, NK cells, CD5+B-1 cells, and subsets of dendriticcells. As disclosed herein, CD11b is also expressed on solid tumor cellsand targeting it leads to anti-tumor effects. Exemplary relevantsequences for CD11b can be found at Accession Nos. NP_001139280.1,NP_000623.2, XP_011544153.1, XP_011544152.1, XP_006721108.1, AAH99660.1,and AH004143.2. In particular embodiments, CD11b refers to SEQ ID NO: 1(FIG. 23).

CD18 is the integrin β chain β2. In humans, a lack of CD18 causesLeukocyte Adhesion Deficiency, a disease defined by a lack of leukocyteextravasation from blood into tissues. Exemplary relevant sequences forCD18 can be found at Accession Nos. AAE18916.1, ADS63144.1, AJL09630.1,AAB21332.1, AAB21402.1, AAB21403.1, and AAB21404.1. In particularembodiments, CD18 refers to SEQ ID NO: 2 (FIG. 24).

MPO is most abundantly expressed in neutrophil granulocytes (a subtypeof white blood cells), and produces hypohalous acids to carry out theirantimicrobial activity. It is a lysosomal protein stored in azurophilicgranules of the neutrophil and released into the extracellular spaceduring degranulation. MPO has a heme pigment, which causes its greencolor in secretions rich in neutrophils, such as pus and some forms ofmucus. MPO is also known to play a prominent role in fibrosis.

Integrins are integral cell-surface transmembrane receptor proteinscomposed of an a chain and a β chain. A given α or β chain may combinewith multiple partners resulting in different integrins. For example, β2combines with the αL chain to form the integrin LFA-1, and combines withthe α M chain to form the integrin Mac-1. Integrins are involved in celladhesion and recognition in various processes including embryogenesis,hemostasis, tissue repair, immune response, and metastatic diffusion oftumor cells.

In humans, the integrin αV protein (CD51) is encoded by the ITGAV gene.CD51 includes disulfide-linked heavy and light chains. CD51 combineswith other β chains to form different integrins. CD51 associates withβ1, β3, β5, β6, and β8 chains to form receptors for vitronectin,fibrinogen, fibronectin, osteopontin, cytotactin, laminin, matrixmetalloproteinase-2, osteomodulin, prothrombin, thrombospondin, and VonWillebrand Factor. Generally, these receptors recognize the R-G-Dsequence in various ligands. Exemplary mammalian sequences encodingintegrin αV can be found at Accession Nos. P06756.2, EDL27275.1, andNP_001290501.1.

Integrin β1 (CD29) is the most abundantly expressed β integrin andassociates with 10 different integrin α subunits. In humans, CD29 isencoded by the ITGB1 gene. CD29 exists in different isoforms viaalternative splicing. There are six alternatively spliced variants forthe gene which encodes 5 proteins with alternate C-termini.

Like other integrins, CD29 has a large extracellular domain and a shortintracellular domain. The cytoplasmic domain of integrin β1 binds to theactin cytoskeleton. Accordingly, integrins link the actin cytoskeletonwith the extracellular matrix and transmit signals bidirectionallybetween the extracellular matrix and cytoplasmic domains. β-integrinstarget integrins to the appropriate subcellular locations, which inadhesive cells is mainly the focal adhesions.

There are three novel isoforms of integrin β1: β1B, β1C, and β1D. Theβ1B isoform inhibits cell adhesion, while the β1C isoform inhibits DNAsynthesis in the G1 phase of cell cycle. The third isoform, β1 D, is astriated muscle-specific isoform. Exemplary mammalian sequences encodingCD29 can be found at Accession Nos. P05556, EDL11832.1, and NP_058718.2.

CD44 is a receptor for hyaluronic acid and can also interact with otherligands, such as osteopontin, collagens, and matrix metalloproteinases(MMPs). CD44 is a multistructural and multifunctional cell surfacemolecule involved in cell proliferation, cell differentiation, cellmigration, angiogenesis, presentation of cytokines, chemokines, andgrowth factors to the corresponding receptors, and docking of proteasesat the cell membrane, as well as in signaling for cell survival. Allthese biological properties are essential to the physiologicalactivities of normal cells, but they are also associated with thepathologic activities of cancer cells.

CD117 is a cytokine receptor expressed on the surface of hematopoieticstem cells as well as other cell types. Altered forms of this receptormay be associated with some types of cancer. CD117 is a receptortyrosine kinase type Ill, which binds to stem cell factor (a substancethat causes certain types of cells to grow), also known as “steelfactor” or “c-kit ligand”. When this receptor binds to stem cell factor(SCF) it forms a dimer that activates its intrinsic tyrosine kinaseactivity that in turn phosphorylates and activates signal transductionmolecules that propagate the signal in the cell. Signaling through CD117μlays a role in cell survival, proliferation, and differentiation.

Before the current disclosure, it was not known that anti-CD11b and/oranti-CD18 (CD11b/CD18) compounds would lead to solid tumor cell death.Anti-CD11b and/or anti-CD18 compounds inhibit, suppress and/or eliminatethe natural physiological activities of their target, such as CD11b/CD18in a solid tumor cell, such that an anti-solid tumor effect (describedbelow) is achieved. In particular embodiments, active agent(s)competitively bind to and/or block CD11b/CD18. Because CD11b forms aheterodimer with CD18, targeting CD11b or CD18 can block the heterodimeras well as the individual proteins.

Moreover, MPO binds to CD11b and is an important mechanism indevelopment of ovarian cancer as well as chemoresistance. Reducing orpreventing this binding using an anti-MPO and/or CD11b antagonist (e.g.,antibody) causes growth arrest and/or apoptosis. This binding reductionor prevention can augment current therapies to the cancer and bypassmechanisms of resistance.

The current disclosure also includes use of anti-Integrin αV oranti-Integrin β1 (collectively Integrin αVβ1) alone or in combinationwith anti-CD11b/CD18 and/or anti-MPO compounds. Anti-αV or anti-β1compounds inhibit, suppress and/or eliminate the natural physiologicalactivities of Integrin αV and/or β1 in a solid tumor cell, such that ananti-solid tumor effect (described below) is achieved. In particularembodiments, active agent(s) competitively bind to and/or block IntegrinαVβ1. Because Integrin αV forms a heterodimer with Integrin β1,targeting Integrin αV or β1 can block the heterodimer as well as theindividual proteins.

Any compound capable of inhibiting, suppressing and/or eliminatingCD11b/CD18, MPO, and/or Integrin αVβ1 activity, among other compoundsdisclosed herein, can be used with the compositions and methodsdisclosed herein. Such compounds are referred to as active agent(s).Active agent(s) can include, for example, proteins, peptides,antibodies, small molecules, fusion proteins, and conjugates, as well asphysiologically acceptable salts, prodrugs, variants, modifications,D-substituted analogs, homologues and allelic variants thereof.

Particularly useful active agent(s) utilize anti-CD11b antibodies (e.g.,rat anti-mouse CD11b available from BD Bioscience, #554980; mouseanti-human CD11b ICRF44, available from Affymetrix; and mouse and humanreactive anti-CD11b M1/10, available from Abcam), anti-CD18 antibodies(e.g., mouse anti-human CD18 TS1/18, available from BioLegend and mouseanti-human CD18 MEM/148, available from Abcam), anti-MPO antibodies(e.g., mouse anti-human MPO available from ThermoFisher Scientific,#MA1-34067, #GM4192, #MA5-15480), anti-integrin αV antibodies (e.g.,mouse anti-human integrin αV available from Santa Cruz Biotechnology#SC-9969 and mouse anti-human integrin αV 272-17E6, available fromAbcam), anti-integrin β1 antibodies (e.g., mouse anti-human integrin β1available from Santa Cruz Biotechnology #SC-37443 and rabbit anti-humanintegrin β1 EP2041Y, available from Abcam), Abciximab, NIF protein,human cathelicidin peptide LL-37 (LL-37), Clopidogrel (Plavix®,Sanofi-Aventis, Paris, France), Cilostazol (Pletal®, OtsukaPharmaceutical, Osaka, Japan), compounds of Formula I, Intetumumab,Abituzumab, or a combination thereof although other compounds thatinhibit, suppress and/or eliminate CD11b/CD18, MPO, and/or Integrin αVβ1can also be used.

Abciximab (previously known as c7E3 Fab, Accession No. DB00054(BIOD00041, BTD00041)) is a glycoprotein IIb/IIIa receptor antagonistcurrently distributed under the trade name ReoPro® (Eli Lilly & Co.,Indianapolis, Ind.). Abciximab is currently used as a plateletaggregation inhibitor mainly used during and after coronary arteryprocedures like angioplasty to prevent platelets from sticking togetherand causing thrombus (blood clot) formation within the coronary artery.Abciximab is indicated for use in individuals undergoing percutaneouscoronary intervention (angioplasty with or without stent placement).

Abciximab has the protein chemical formula C₆₄₆₂H₉₉₆₄N₁₆₉₀O₂₀₄₉S₄₈ and aweight of 145651 D. Exemplary Abciximab sequences include heavy chain 1(FIG. 25A; SEQ ID NO: 3); light chain 1 (FIG. 25B; SEQ ID NO: 4); heavychain 2 (FIG. 25C; SEQ ID NO: 5); and light chain 2 (FIG. 25D; SEQ IDNO: 6).

NIF is a 41-kDa CD11/CD18 P(2) integrin-binding protein isolated fromthe canine hookworm (Ancylostoma caninum). NIF includes 257 amino acidsand contains seven glycosylation sites. It blocks PMN adhesion in aconcentration-dependent manner with, in particular embodiments, acomplete blockade occurring at 10 nM NIF. NIF can effectively inhibitneutrophil activities including adhesion to endothelial cells, releaseof hydrogen peroxide and superoxide ions, as well as chemotaxis,aggregation and phagocytosis of neutrophil, etc (Moyle et al: J Biolchem 1994, 269(13):10008-10015). Nucleic acid sequences encoding NIF canbe found at, for example, Accession Nos. L27427.1, DI138516.1,DI132333.1, DI127964.1, DD041939.1, DD041938.1, and DD041937.1. FIG. 26provides an exemplary encoding sequence (SEQ ID NO: 7).

Human cathelicidin peptide LL-37 (LL-37) includes the C-terminal 37amino acids of human cathelicidin (e.g.,LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 8)). LL-37 wasoriginally referred to as FALL39, named for the first 4 N-terminal aminoacids of this domain and the total number of residues (i.e., 39). LL-37is a peptide predicted to contain an amphipathic α helix and lackscysteine, making it different from all other previously isolated humanpeptide antibiotics of the defensin family, each of which contain 3disulfide bridges. Full length human cathelicidin includes thecathelin-like precursor protein and the C-terminal LL-37 peptide, thusincluding 170 amino acids.

Cathelicidins more generally are a family of endogenous antimicrobialpeptides which form a part of the innate immunity that protects the hostfrom infection (Eckmann, Gastroenterol. 2005; 21(2):147-51).Cathelicidin exists in human as LL-37 and in mice as mCRAMP (Gudmundssonet al., Eur J Biochem. 1996; 238(2):325-32; Gallo et al., J Biol Chem.1997; 272(20):13088-93). Cathelicidin is secreted from the apicalsurface that is facing exterior environment such as intestine (Schauberet al., Eur J Gastroenterol Hepatol. 2006; 18(6):615-21) and salivarygland (Murakami et al., J Dent Res. 2002; 81(12):845-50) by epithelialcells (Schauber et al., Eur J Gastroenterol Hepatol. 2006; 18(6):615-21)and immune cells such as macrophages (Koon et al., Gastroenterology.2011; 141(5):1852-63 e1-3).

Intetumumab, also known as CNTO 95, is the first fully human monoclonalantibody that recognizes all the members of the CD51 family ofintegrins. Intetumumab binds CD51 integrins with high affinity andspecificity.

Abituzumab is a recombinant humanized monoclonal antibody for humanCD51. It is used to inhibit CD51 expressed on CRPC cells, tumor vesselsand osteoclasts involved in bone metastasis. Abituzumab is in phase IIclinical trials for metastatic castration-resistant prostate cancer(CRPC).

Various polyclonal and monoclonal antibodies against CD29 arecommercially available.

While exemplary protein and nucleic acid sequences are provided, one ofordinary skill in the art understands that additional supportingsequences may be found in publicly available databases. Protein and/ornucleic acid sequences for use in the compositions and methods disclosedherein also include proteins and/or nucleic acid sequences having atleast 90% sequence identity to a protein or nucleic acid disclosedherein; at least 91% sequence identity to a protein or nucleic aciddisclosed herein; at least 92% sequence identity to a protein or nucleicacid disclosed herein; at least 93% sequence identity to a protein ornucleic acid disclosed herein; at least 94% sequence identity to aprotein or nucleic acid disclosed herein; at least 95% sequence identityto a protein or nucleic acid disclosed herein; at least 96% sequenceidentity to a protein or nucleic acid disclosed herein; at least 97%sequence identity to protein or nucleic acid disclosed herein; at least98% sequence identity to a protein or nucleic acid disclosed herein; orat least 99% sequence identity to a protein or nucleic acid disclosedherein.

“% sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweensequences as determined by the match between strings of such sequences.“Identity” (often referred to as “similarity”) can be readily calculatedby known methods, including those described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, N Y (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, N Y (1994); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, N J (1994);Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Oxford University Press, NY (1992). Preferred methods todetermine sequence identity are designed to give the best match betweenthe sequences tested. Methods to determine sequence identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR, Inc., Madison, Wis.). Multiple alignment of thesequences can also be performed using the Clustal method of alignment(Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also includethe GCG suite of programs (Wisconsin Package Version 9.0, GeneticsComputer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul,et al., J. Mol. Biol. 215, 403-410 (1990); DNASTAR (DNASTAR, Inc.,Madison, Wis.); and the FASTA program incorporating the Smith-Watermanalgorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.](1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher:Plenum, New York, N.Y.). Within the context of this disclosure it willbe understood that where sequence analysis software is used foranalysis, the results of the analysis are based on the “default values”of the program referenced. “Default values” mean any set of values orparameters which originally load with the software when firstinitialized.

Reference to proteins described herein also include variants,modifications, D-substituted analogs, homologues and allelic variantsthereof. “Variants” of proteins disclosed herein include proteins havingone or more amino acid additions, deletions, stop positions, orsubstitutions, as compared to a protein disclosed herein.

An amino acid substitution can be a conservative or a non-conservativesubstitution. Variants of proteins disclosed herein can include thosehaving one or more conservative amino acid substitutions. A“conservative substitution” involves a substitution found in one of thefollowing conservative substitutions groups: Group 1: alanine (Ala orA), glycine (Gly or G), Ser, Thr; Group 2: aspartic acid (Asp or D),Glu; Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: Arg,lysine (Lys or K), histidine (His or H); Group 5: lie, leucine (Leu orL), methionine (Met or M), valine (Val or V); and Group 6: Phe, Tyr,Trp.

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function, chemical structure, or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cys; acidic: Asp, Glu, Asn, and Gln; smallaliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, andGly; polar, negatively charged residues and their amides: Asp, Asn, Glu,and Gln; polar, positively charged residues: His, Arg, and Lys; largealiphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and largearomatic residues: Phe, Tyr, and Trp. Additional information is found inCreighton (1984) Proteins, W.H. Freeman and Company.

Substitutions can be made to improve the pharmacokinetic and/orpharmacodynamic (PK/PD) properties of a protein. For example, residuesthat are sensitive to degradation (e.g., Met) can be replaced orsubstituted with residues that are less sensitive to degradation (e.g.,Nle). Substitution of the C-terminal acid with an amide can also impartstability. An anionic moiety substitution at the N-terminus can alsoincrease protein stability.

Modifications, as used herein, include altering amino acid structures orattaching functional groups to amino acids to improve, for example,PK/PD properties. In particular embodiments, use of nonhydrolyzablephosphate substitutions can impart a stabilizing effect on the phosphategroups, and can provide stability against phosphatase enzymes. Organicor inorganic chemical entities can also be attached to proteins.

Non-limiting examples of appropriate chemical entities includeL-p-phosphonomethyl-phenylalanine (Pmp) (OH2); D-Pmp(OH2); D-Pmp(OHEt);Pmp(Et2); D-Pmp(Et2); L-Tyr L-Tyr(PO3H2) (p-phospho-Tyrosine);L-Phe(p-NH2); L-Phe(p-CO2H); L-Aspartate; D-Aspartate; L-Glutamate; andD-Glutamate. Pmp can be substituted with (p-phosphatityl-phenylalanine)(Ppa); (p-Phosphono(difluoro-methyl)-Phenylalanine) (Pfp) or(p-Phosphono-methylketo-Phenylalanine) (Pkp). Exemplary chemicalentities can be attached by way of a linker, such as anaminoethyloxyethyloxy-acetyl linker or by any other suitable means.

PEGylation provides another potential modification. PEGylation is aprocess by which polyethylene glycol (PEG) polymer chains are covalentlyconjugated to other molecules such as drugs or proteins. Several methodsof PEGylating proteins have been reported in the literature. Forexample, N-hydroxy succinimide (NHS)-PEG was used to PEGylate the freeamine groups of lysine residues and N-terminus of proteins; PEGs bearingaldehyde groups have been used to PEGylate the amino-termini of proteinsin the presence of a reducing reagent; PEGs with maleimide functionalgroups have been used for selectively PEGylating the free thiol groupsof cysteine residues in proteins; and site-specific PEGylation ofacetyl-phenylalanine residues can be performed.

Covalent attachment of proteins to PEG has proven to be a useful methodto increase the circulating half-lives of proteins in the body(Abuchowski, et al., Cancer Biochem. Biophys., 1984, 7:175-186;Hershfield, et al., N. Engl. J. Medicine, 1987, 316:589-596; and Meyers,et al., Clin. Pharmacol. Ther., 1991, 49:307-313). The attachment of PEGto proteins not only protects the molecules against enzymaticdegradation, but also reduces their clearance rate from the body. Thesize of PEG attached to a protein has significant impact on thecirculating half-life of the protein. The ability of PEGylation todecrease clearance is generally not a function of how many PEG groupsare attached to the protein, but the overall molecular weight of thealtered protein. PEGylation decreases the rate of clearance from thebloodstream by increasing the apparent molecular weight of the molecule.Up to a certain size, the rate of glomerular filtration of proteins isinversely proportional to the size of the protein. Usually the largerthe PEG is, the longer the in vivo half-life of the attached protein is.In addition, PEGylation can also decrease protein aggregation (Suzuki etal., Biochem. Bioph. Acta vol. 788, pg. 248 (1984)), alter proteinimmunogenicity (Abuchowski et al.; J. Biol. Chem. vol. 252 μg. 3582(1977)), and increase protein solubility as described, for example, inPCT Publication No. WO 92/16221).

Several sizes of PEGs are commercially available (Nektar AdvancedPEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7.1), which aresuitable for producing proteins with targeted circulating half-lives. Avariety of active PEGs have been used including mPEG succinimidylsuccinate, mPEG succinimidyl carbonate, and PEG aldehydes, such asmPEG-propionaldehyde.

“D-substituted analogs” include proteins disclosed herein having onemore L-amino acids substituted with one or more D-amino acids. TheD-amino acid can be the same amino acid type as that found in thereference sequence or can be a different amino acid. Accordingly,D-analogs can also be variants.

Clopidogrel (marketed as Plavix® by Sanofi (Paris France)) andCilostazol (marketed as Pletal) by Otsuka Pharmaceuticals Co. (Osaka,JP)) share a common mechanism of action with Abciximab. Thus, thesecompounds are also appropriate for use in the compositions and treatmentmethods disclosed herein. More particularly, Clopidogrel is an oralantiplatelet agent used to inhibit blood clots in various diseases suchas coronary artery disease, peripheral vascular disease, cerebrovasculardisease and to prevent myocardial infarction. It is an irreversibleinhibitor of P2Y12 adenosine diphosphate receptor found on the membranesof platelet cells.

Clopidogrel has a molecular weight of 321.82 g/mol and a molecularformula of C₁₆H₁₆ClNO₂S. Clopidogrel has the following structuralformula,

and the following IUPAC name: methyl(2S)-2-(2-chlorophenyl)-2-(6,7-dihydro-4H-thieno[3,2-c]pyridin-5-yl)acetate.

In particular embodiments, Cilostazol can be used in the compositionsand methods disclosed herein. Cilostazol is a phosphodiesteraseinhibitor with therapeutic focus on cyclic adenosine monophosphate(cAMP). Cilostazol inhibits platelet aggregation and is a directarterial vasodilator. Cilostazol is used to treat intermittentclaudication, a condition caused by narrowing of the arteries thatsupply the legs with blood. Cilostazol alleviates intermittentclaudication by dilating the arteries to improve the flow of blood andoxygen to the legs and by inhibiting the action of phosphodiesteraseIll.

Cilostazol has a molecular weight of 369.46 g/mol and a molecularformula of C₂₀H₂₇N₅O₂. Cilostazol has the following structural formula,

and the following IUPAC name,6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone.

In particular embodiments, a compound of formula (I) can be used in thecompositions and methods disclosed herein. Formula (I) includes

wherein:B is absent and R1 is phenyl; orB is methylene and R1 is phenyl or phenyl substituted with one fluoro;N is nitrogen;X is selected from the group including O and S; andR3 is selected from the group including 4-carboxyphenyl and3-carboxy-4-chlorophenyl. In embodiments, the compound is asubstantially pure single Z conformer. In certain embodiments, thecompound is

Methods disclosed herein include treating subjects (humans, veterinaryanimals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle,goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice,fish, etc.)) with active agent(s) disclosed herein. Subjects in need ofa treatment (in need thereof) are subjects having a solid tumor, whetherthe solid tumor occurred naturally or by induction through a researchprotocol. Subjects in need thereof also include those who have had asolid tumor in the past.

Treating subjects includes delivering therapeutically effective amounts.Therapeutically effective amounts include those that provide effectiveamounts, prophylactic treatments, and/or therapeutic treatments.

An “effective amount” is the amount of active agent(s) or composition(s)necessary to result in a desired physiological change in a subject.Effective amounts are often administered for research purposes.Effective amounts disclosed herein have an anti-solid tumor effect.

A “prophylactic treatment” includes a treatment administered to asubject who does not display signs or symptoms of distant or metastaticsolid tumors or chemoresistance, or displays only early signs orsymptoms of distant or metastatic solid tumors or chemoresistance suchthat treatment is administered for the purpose of diminishing,preventing, or decreasing the risk of developing further distant ormetastatic solid tumors or chemoresistance. Thus, a prophylactictreatment functions as a preventative treatment against the developmentof distant or metastatic solid tumors or chemoresistance further.

A “therapeutic treatment” includes a treatment administered to a subjectwho has one or more solid tumor(s) or who has had one or more solidtumor(s) and is administered to the subject for the purpose of providingan anti-solid tumor effect. Therapeutic treatments can be distinguishedfrom effective amounts based on the presence or absence of a researchcomponent to the administration. As will be understood by one ofordinary skill in the art, however, in human clinical trials therapeutictreatments and effective amounts can overlap.

An anti-solid tumor effect means a decrease in solid tumor volume, adecrease in the number of solid tumor cells, a decrease in the number ofsolid tumors, slowed or inhibited growth of a solid tumor, induction ofapoptosis in solid tumor cells, a decrease in solid tumor metastasesand/or a delay in the development of chemoresistance. Solid tumor cellscan include cancer cells, chemoresistant cells, cancer stem cells, andany other cells found within benign, pre-malignant or malignant tumors.Solid tumor cells can also include any tumor cells left behind aftersurgical removal or another treatment that may lead to a tumor or cancerrecurrence if left untreated.

In particular embodiments, an anti-solid tumor effect can lead to ananti-cancer effect. An anti-cancer effect can be manifested by adecrease in cancer recurrence, an increase in time before cancerrecurrence, an increase in life expectancy, an increase in lifeduration, or a decrease in various physiological symptoms associatedwith a solid tumor cancerous condition.

Solid tumors that can be treated with the compositions and methodsdisclosed herein include benign tumors, pre-malignant tumors, malignanttumors, and chemoresistant tumors. The compositions and methodsdisclosed herein can also be used to treat chemoresistant cancer cellsand/or cancer stem cells whether part of or independent from a solidtumor at the time of treatment.

Benign tumors include fibroid tissue solid tumors, such as uterinefibroids.

Pre-malignant or malignant solid tumors include those found in subjectswith acoustic neuroma, adenocarcinoma, astrocytoma, basal cell cancer,bile duct cancer, bladder cancer, brain cancer, breast cancer,bronchogenic cancer, central nervous system cancer, cervical cancer,chondrosarcoma, choriocarcinoma, colon cancer, craniopharyngioma,ependymoma, Ewing's tumor, fibrosarcoma, glandular cancer, glioma,hemangioblastoma, hepatocellular carcinoma, hepatoma, kidney cancer,leiomyosarcoma, liver cancer, liposarcoma, lung cancer, melanoma,medulloblastoma, medullary cancer, medullary thyroid cancer, menangioma,mesothelioma, myxosarcoma, neuroblastoma, oligodendroglioma, osteogenicsarcoma, ovarian cancer, papillary adenocarcinomas, papillary thyroidcancer, pancreatic cancer, pheochromocytomas papillary cancer, pinealcancer, prostate cancer, renal cell cancer, retinoblastoma,rhabdomyosarcoma, sarcoma, sebaceous gland cancer, seminoma, skincancer, squamous cell cancer, sweat gland cancer, synovioma, testicularcancer, and/or Wilms' tumor. Solid tumors do not include cancersassociated with blood or other liquid cancers.

Chemoresistant cancer cells are cancer cells that have become lesssusceptible to the therapeutic effects of a chemotherapeutic agent.Chemoresistance can be identified using in vitro assays. For example,the overexpression of Toll Like Receptor 4 (TLR-4) in human tumorscorrelates with chemoresistance and metastasis. Therefore, detectingoverexpression of TLR-4 in a cancer cell after exposure to thechemotherapeutic agent indicates chemoresistance. Chemoresistance canalso be identified by a reduction in the therapeutic effect of anadministered treatment. In particular embodiments, therapeuticallyeffective amounts kill chemoresistant cancer cells.

Cancer stem cells possess the characteristics associated with normalstem cells, such as self-renewal and differentiation. Thus, they havethe ability to give rise to all cell types found in a particular cancersample. Cancer stem cells may generate tumors through the stem cellprocesses of self-renewal and differentiation into multiple cell types.Such cells are hypothesized to persist in subjects as a distinctpopulation and cause relapse and metastasis by giving rise to newtumors, even after successful initial treatment. In particularembodiments, therapeutically effective amounts kill cancer stem cells.

For administration, therapeutically effective amounts (also referred toherein as doses) can be initially estimated based on results from invitro assays and/or animal model studies. Such information can be usedto more accurately determine useful doses in subjects of interest. Invitro assays can assess cell growth as determined by MTT and colonyformation assays. Cell counting as a golden standard can be performedroutinely to determine cell doubling times and growth rates.

The actual dose amount administered to a particular subject can bedetermined by a physician, veterinarian, or researcher taking intoaccount parameters such as physical, physiological and psychologicalfactors including target, body weight, type of tumor, size of tumor,location of tumor, stage of cancer, type of cancer, previous orconcurrent therapeutic interventions, idiopathy of the subject, and/orroute of administration.

Exemplary doses can include 0.05 mg/kg to 5.0 mg/kg of the activeagent(s). For certain indications, the total daily dose can be 0.05mg/kg to 30.0 mg/kg active agent(s) administered to a subject one tothree times a day, including administration of total daily doses of0.05-3.0, 0.1-3.0, 0.5-3.0, 1.0-3.0, 1.5-3.0, 2.0-3.0, 2.5-3.0, and0.5-3.0 mg/kg/day of administration forms of the active agent(s) using60-minute oral, intravenous or other dosing. In one particular example,doses can be administered QD or BID to a subject with, e.g., total dailydoses of 1.5 mg/kg, 3.0 mg/kg, or 4.0 mg/kg of a composition with up to92-98% wt/v of the active agent(s).

Additional useful doses can often range from 0.1 to 5 μg/kg or from 0.5to 1 μg/kg. In other examples, a dose can include 1 μg/kg, 5 μg/kg, 10μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45μg/kg, 50 μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 pg/kg, 80μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, 150 μg/kg, 200 μg/kg,250 μg/kg, 350 pg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600μg/kg, 650 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 850 μg/kg, 900 μg/kg,950 μg/kg, 1000 μg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In otherexamples, a dose can include 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg,400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000mg/kg, or more.

Particularly useful doses can include 0.25-5 mg/kg intravenous bolusfollowed by a continuous intravenous infusion of 0.125-2.5 μg/kg/min for12 hours, per treatment.

Doses referred to herein can include one or more active agent(s)collectively or individually.

Therapeutically effective amounts can be achieved by administeringsingle or multiple doses during the course of a treatment regimen (e.g.,hourly, every 2 hours, every 3 hours, every 4 hours, every 6 hours,every 9 hours, every 12 hours, every 18 hours, daily, every other day,every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2weeks, every 3 weeks, or monthly).

One or more active agent(s) can be administered simultaneously or withina selected time window, such as within 10 minutes, 1 hour, 3 hour, 10hour, 15 hour, 24 hour, or 48 hour time windows or when thecomplementary active agent(s) is within a clinically-relevanttherapeutic window.

In particular embodiments, the active agent(s) can be used inconjunction with other cancer treatments such as agonadotropin-releasing hormone agonist or antagonist (e.g., Lupron,Zoladex (Goserelin), Degarelix, Ozarelix, ABT-620 (Elagolix), TAK-385(Relugolix), EP-100 or KLH-2109); a phosphoinositide 3-kinase (PI3K)inhibitor, a TORC inhibitor, or a dual PI3K/TORC inhibitor (e.g.,BEZ-235, BKM120, BGT226, BYL-719, GDC0068, GDC-0980, GDC0941, GDC0032,MK-2206, OSI-027, CC-223, AZD8055, SAR245408, SAR245409, PF04691502,WYE125132, GSK2126458, GSK-2636771, BAY806946, PF-05212384, SF1126,PX866, AMG319, ZSTK474, Call01, PWT33597, LY-317615 (enzastaurinhydrochloride), CU-906, or CUDC-907); a CYP17 inhibitor (e.g.,abiraterone acetate (Zytiga), TAK-700 (orteronel), or VT-464);prednisone; an osteoprotective agent; a radiation therapy; a kinaseinhibitor (e.g. MET, VEGFR, EGFR, MEK, SRC, AKT, RAF, FGFR, CDK4/6);Provenge, Prostvac, Ipilimumab, a PD-1 inhibitor; a taxane or tubulininhibitor; an anti-STEAP-1 antibody; a heat shock protein 90 (HSP90) orheat shock protein 27 (HSP27) pathway modulator; an anti-androgen (e.g.bicalutamide); and/or immunotherapy.

The active agent(s) also can be administered with olaparib, bevacizumab,albumin bound paclitaxel, altretamine, capecitabine, cyclophosphamide,etoposide, gemcitabine, ifosfamide, irinotecan, liposomal doxorubicin,melphalan, pemetrexed, topotecan, or vinorelbine.

In particular embodiments, the active agent(s) are administered withcisplatin and/or docetaxel.

Active agent(s) can also be administered with anesthetics includingethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine,mepivacaine, procaine, ropivacaine, tetracaine, desflurane, isoflurane,ketamine, propofol, sevoflurane, codeine, fentanyl, hydromorphone,marcaine, meperidine, methadone, morphine, oxycodone, remifentanil,sufentanil, butorphanol, nalbuphine, tramadol, benzocaine, dibucaine,ethyl chloride, xylocaine, and/or phenazopyridine.

Combination therapies refer to those situations in which two or moredifferent active agent(s) are administered in overlapping regimens sothat the subject is simultaneously exposed to both agents intherapeutically effective amounts.

Methods disclosed herein can further include determining whether asubject has developed chemoresistance to first-line or second-linechemotherapeutic agents prior to administering a therapeuticallyeffective amount. In particular embodiments, the disclosed compositionsand methods can be utilized when chemoresistant cancer cells aredetected in a subject. In particular embodiments, the disclosedcompositions and methods can be utilized before chemoresistant cancercells are detected in a subject.

As suggested, the one or more active agent(s) can be formulated into acomposition for administration. Compositions include at least one activeagent and at least one pharmaceutically-acceptable carrier and/orexcipient.

For injection, compositions can be formulated as aqueous solutions, suchas in buffers including Hanks' solution, Ringer's solution, orphysiological saline. The aqueous solutions can contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.Examples of suitable aqueous and non-aqueous carriers, which may beemployed in the injectable formulations include water, ethanol, polyols(such as glycerol, propylene glycol, polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyloleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of selected particle size in the case ofdispersions, and by the use of surfactants.

Particular embodiments can be clear, colorless, sterile, non-pyrogenicsolutions for intravenous (IV) use. Single use vials containing 2 mg/mLof Abciximab and/or NIF in a buffered solution (pH7.2) of 0.01 M sodiumphosphate, 0.15 M sodium chloride and 0.001% polysorbate 80 in Water forInjection can be provided. In particular embodiments of thisformulation, no preservatives are added.

Injectable formulations may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like in the compositions.

Alternatively, the composition can be in lyophilized form and/orprovided in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. Lyophilized compositions caninclude less than 5% water content; less than 4.0% water content; orless than 3.5% water content.

In particular embodiments, the composition can be in a unit dosage form,such as in a suitable diluent in sterile, hermetically sealed ampoulesor sterile syringes.

In particular embodiments, in order to prolong the effect of acomposition, it is desirable to slow the absorption of the activeagent(s) following injection. Compositions can be formulated assustained-release systems utilizing semipermeable matrices of solidpolymers containing at least one administration form. Varioussustained-release materials have been established and are well known bythose of ordinary skill in the art. Sustained-release systems may,depending on their chemical nature, release agents followingadministration for a few weeks up to over 100 days.

In particular embodiments, delayed absorption can be accomplished bydissolving or suspending the active agent(s) in an oil vehicle. Inparticular embodiments, administration forms can be formulated as depotpreparations. Depot preparations can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salts. In addition,prolonged absorption of the injectable composition may be brought aboutby the inclusion of active agent(s) which delay absorption such asaluminum monostearate and gelatin.

Injectable depot forms can be made by forming microencapsule matrices ofadministration forms in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of administration formto polymer, and the nature of the particular polymer employed, the rateof administration form release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Injectable depot formulations are also prepared by entrapping the activeagent(s) in liposomes or microemulsions which are compatible with bodytissue.

Alternatively, delayed absorption of a composition can be accomplishedby the use of a liquid suspension of crystalline or amorphous materialhaving poor water solubility. The rate of absorption of the activeagent(s) then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form.

Compositions can also be formulated for oral administration. Foringestion, compositions can take the form of tablets, pills, lozenges,sprays, liquids, and capsules formulated in conventional manners.Ingestible compositions can be prepared using conventional methods andmaterials known in the pharmaceutical art. For example, U.S. Pat. Nos.5,215,754 and 4,374,082 relate to methods for preparing swallowablecompositions. U.S. Pat. No. 6,495,177 relates to methods to preparechewable supplements with improved mouthfeel. U.S. Pat. No. 5,965,162,relates to compositions and methods for preparing comestible units whichdisintegrate quickly in the mouth.

Ingestible compositions may have a shape containing no sharp edges and asmooth, uniform and substantially bubble free outer coating. Coatings ofingestible compositions can be derived from a polymeric film. Such filmcoatings reduce the adhesion of the compositions to the inner surface ofthe mouth and can aid in masking potential unpleasant tastes. Coatingscan also protect the compositions from atmospheric degradation.Exemplary polymeric films include vinyl polymers, cellulosics, acrylatesand methacrylates, natural gums and resins such as zein, gelatin,shellac and acacia. Other common excipients used in ingestiblecompositions include sucrose, fructose, lactose, glucose, lycasin,xylitol, lactitol, erythritol, mannitol, isomaltose, dextrose,polydextrose, dextrin, compressible cellulose, compressible honey,compressible molasses, fondant or gums, vegetable oils, animal oils,alkyl polysiloxanes, corn starch, potato starch, pre-gelatinizedstarches, stearic acid, calcium stearate, magnesium stearate, zincstearate, benzoic acid, and colorants

For administration by inhalation (e.g., nasal or pulmonary), thecompositions can be formulated as aerosol sprays for pressurized packsor a nebulizer, with the use of suitable propellants, e.g.dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetra-fluoroethane.

As suggested, nanoparticle formulations for a variety of administrationroutes can also be used.

Any composition disclosed herein can advantageously include any otherpharmaceutically acceptable carriers which include those that do notproduce significantly adverse, allergic, or other untoward reactionsthat outweigh the benefit of administration, whether for research,prophylactic, and/or therapeutic treatments. Exemplary pharmaceuticallyacceptable carriers and formulations are disclosed in Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover,formulations can be prepared to meet sterility, pyrogenicity, generalsafety, and purity standards as required by U.S. FDA Office ofBiological Standards and/or other relevant foreign regulatory agencies.

Exemplary generally used pharmaceutically acceptable carriers includeany and all bulking agents or fillers, solvents or co-solvents,dispersion media, coatings, surfactants, antioxidants (e.g., ascorbicacid, methionine, vitamin E), preservatives, isotonic agents, absorptiondelaying agents, salts, stabilizers, buffering agents, chelating agents(e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.Fillers and excipients are commercially available from companies such asAldrich Chemical Co., FMC Corp, Bayer, BASF, Alexi Fres, Witco,Mallinckrodt, Rhodia, ISP, and others.

Active agent(s) include their pharmaceutically acceptable salts and/orpro-drugs. A pharmaceutically acceptable salt includes any salt thatretains the activity of the active agent(s) and is acceptable forpharmaceutical use. A pharmaceutically acceptable salt also refers toany salt which may form in vivo as a result of administration of anacid, another salt, or a prodrug which is converted into an acid orsalt.

Suitable pharmaceutically acceptable acid addition salts can be preparedfrom an inorganic acid or an organic acid. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric and phosphoric acid. Appropriate organic acids can be selectedfrom aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids.

Suitable pharmaceutically acceptable base addition salts includemetallic salts made from aluminum, calcium, lithium, magnesium,potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

A prodrug includes an active agent which is converted to atherapeutically active compound after administration, such as bycleavage of a protein or by hydrolysis of a biologically labile group.

In particular embodiments, the compositions can include, for example, 25μg/mL or mg-5 mg/mL or mg, 50 μg/mL or mg-5 mg/mL or mg, 100 μg/mL ormg-5 mg/mL or mg, 150 μg/mL or mg-5 mg/mL or mg, 200 μg/mL or mg-5 mg/mLor mg, 250 μg/mL or mg-5 mg/mL or mg, 300 μg/mL or mg-5 mg/mL or mg, 350μg/mL or mg-5 mg/mL or mg, 400 μg/mL or mg-5 mg/mL or mg, 450 μg/mL ormg-5 mg/mL or mg, 500 μg/mL or mg-5 mg/mL or mg, 550 μg/mL or mg-5 mg/mLor mg, 600 μg/mL or mg-5 mg/mL or mg, 650 μg/mL or mg-5 mg/mL or mg, 700μg/mL or mg-5 mg/mL or mg, 750 μg/mL or mg-5 mg/mL or mg, 800 μg/mL ormg-5 mg/mL or mg, 850 μg/mL or mg-5 mg/mL or mg, 900 μg/mL or mg-5 mg/mLor mg, 950 μg/mL or mg-5 mg/mL or mg, 1 mg/mL or mg-5 mg/mL or mg, 1.5mg/mL or mg-5 mg/mL or mg, 2 mg/mL or mg-5 mg/mL or mg, 2.5 mg/mL ormg-5 mg/mL or mg, 3 mg/mL or mg-5 mg/mL or mg, 3.5 mg/mL or mg-5 mg/mLor mg, 4 mg/mL or mg-5 mg/mL or mg, 4.5 mg/mL or mg-5 mg/mL or mg, 25μg/mL or mg-2.5 mg/mL or mg, 50 μg/mL or mg-2.5 mg/mL or mg, 100 μg/mLor mg-2.5 mg/mL or mg, 150 μg/mL or mg-2.5 mg/mL or mg, 200 μg/mL ormg-2.5 mg/mL or mg, 250 μg/mL or mg-2.5 mg/mL or mg, 300 μg/mL or mg-2.5mg/mL or mg, 350 μg/mL or mg-2.5 mg/mL or mg, 400 μg/mL or mg-2.5 mg/mLor mg, 450 μg/mL or mg-2.5 mg/mL or mg, 500 μg/mL or mg-2.5 mg/mL or mg,550 μg/mL or mg-2.5 mg/mL or mg, 600 μg/mL or mg-2.5 mg/mL or mg, 650μg/mL or mg-2.5 mg/mL or mg, 700 μg/mL or mg-2.5 mg/mL or mg, 750 μg/mLor mg-2.5 mg/mL or mg, 800 μg/mL or mg-2.5 mg/mL or mg, 850 μg/mL ormg-2.5 mg/mL or mg, 900 μg/mL or mg-2.5 mg/mL or mg, 950 μg/mL or mg-2.5mg/mL or mg, 1 mg/mL or mg-2.5 mg/mL or mg, 1.5 mg/mL or mg-2.5 mg/mL ormg, 2 mg/mL or mg-2.5 mg/mL or mg, 25 μg/mL or mg-1 mg/mL or mg, 50μg/mL or mg-1 mg/mL or mg, 100 μg/mL or mg-1 mg/mL or mg, 150 μg/mL ormg-1 mg/mL or mg, 200 μg/mL or mg-1 mg/mL or mg, 250 μg/mL or mg-1 mg/mLor mg, 300 μg/mL or mg-1 mg/mL or mg, 350 μg/mL or mg-1 mg/mL or mg, 400μg/mL or mg-1 mg/mL or mg, 450 μg/mL or mg-1 mg/mL or mg, 500 μg/mL ormg-1 mg/mL or mg, 550 μg/mL or mg-1 mg/mL or mg, 600 μg/mL or mg-1 mg/mLor mg, 650 μg/mL or mg-1 mg/mL or mg, 700 μg/mL or mg-1 mg/mL or mg, 750μg/mL or mg-1 mg/mL or mg, 800 μg/mL or mg-1 mg/mL or mg, 850 μg/mL ormg-1 mg/mL or mg, 900 μg/mL or mg-1 mg/mL or mg, 950 μg/mL or mg-1 mg/mLor mg, 25 μg/mL or mg-750 μg/mL or mg, 50 μg/mL or mg-750 μg/mL or mg,100 μg/mL or mg-750 μg/mL or mg, 150 μg/mL or mg-750 μg/mL or mg, 200μg/mL or mg-750 μg/mL or mg, 250 μg/mL or mg-750 μg/mL or mg, 300 μg/mLor mg-750 μg/mL or mg, 350 μg/mL or mg-750 μg/mL or mg, 400 μg/mL ormg-750 μg/mL or mg, 450 μg/mL or mg-750 μg/mL or mg, 500 μg/mL or mg-750μg/mL or mg, 550 μg/mL or mg-750 μg/mL or mg, 600 μg/mL or mg-750 μg/mLor mg L, 650 μg/mL or mg-750 μg/mL or mg, 700 μg/mL or mg-750 μg/mL ormg, 25 μg/mL or mg-500 μg/mL or mg, 50 μg/mL or mg-500 μg/mL or mg, 100μg/mL or mg-500 μg/mL or mg, 150 μg/mL or mg-500 μg/mL or mg, 200 μg/mLor mg-500 μg/mL or mg, 250 μg/mL or mg-500 μg/mL or mg, 300 μg/mL ormg-500 μg/mL or mg, 350 μg/mL or mg-500 μg/mL or mg, 400 μg/mL or mg-500μg/mL or mg, 450 μg/mL or mg-500 μg/mL or mg, 25 μg/mL or mg-250 μg/mLor mg, 50 μg/mL or mg-250 μg/mL or mg, 100 μg/mL or mg-250 μg/mL or mg,150 μg/mL or mg-250 μg/mL or mg, 200 μg/mL or mg-250 μg/mL or mg, 25μg/mL or mg-100 μg/mL or mg, or 50 μg/mL or mg-100 μg/mL or mg of one ormore of the active agent(s).

In particular embodiments, ratios of active agent(s) can include: (e.g.,Abciximab:NIF (and whether in the same or different compositions)):1:0.0001; 1:0.001; 1:0.005; 1:0.0075; 1:0.01; 1:0.05; 1:0.075; 1:0.1;1:0.5; 1:0.75; 1:1; 1:1.25; 1:1.5; 1:1.75; 1:8; 1:1.2; 1:1.25; 1:1.3;1:1.35; 1:1.4; 1:1.5; 1:1.75; 1:2; 1:3; 1:4; 1:5; 1:6:1:7; 1:8; 1:9;1:10; 1:15; 1:20; 1:30; 1:40; 1:50; 1:60; 1:70; 1:80; 1:90; 1:100;1:200; 1:300; 1:400; 1:500; 1:600; 1:700; 1:800; 1:900; 1:1000. In theseexemplary embodiments, Abciximab and/or NIF can be replaced with otheranti-CD11b antibodies, anti-integrin αV antibodies, anti-integrin β1antibodies, human cathelicidin peptide LL-37, Clopidogrel, Cilostazol,compounds of Formula I, Intetumumab, Abituzumab, or a combinationthereof in all potential combinations.

In particular embodiments, Abciximab:NIF (or other active agent(s)described herein) can be expressed as a fusion protein and includedwithin a composition. Expression of fusion proteins is well known, andfusion proteins can include any appropriate linkers or spacers tofacilitate bioavailability of fused proteins. Proteins can also belinked through any other mechanism known or available to those ofordinary skill in the art. In particular embodiments, fusion proteinsincluding active agent(s) disclosed herein can further include atargeting molecule, an apoptosis inducer or an angiogenesis inhibitor.

Whether alone or in combination, proteins disclosed herein can beadministered as the proteins themselves or as nucleic acids encoding theproteins (whether fused or individually).

Any vector suitable for administering nucleic acid molecules encodingproteins to a solid tumor cell such that the solid tumor cells expressthe proteins may be employed. In particular embodiments, the nucleicacid molecule is incorporated into a viral particle to mediate genetransfer to a solid tumor cell. In particular embodiments, the virussimply can be exposed to the appropriate host cell under physiologicconditions, permitting uptake of the virus. Examples of viral vectorsinclude those derived from adenovirus, adeno-associated virus,retrovirus, lentivirus, herpes simplex virus, vaccinia virus, etc.

In particular embodiments, the nucleic acids or the vectors includingthe nucleic acids can be transfected into cells by ex vivotransformation; injection, such as subcutaneous, intradermal,intramuscular, intravenous, intraperitoneal injection; liposome mediatedtransfection; receptor mediated transfection; etc.

Vectors can include regulatory sequences to control the expression ofthe nucleic acid molecules. These regulatory sequences can be eukaryoticor prokaryotic in nature. In particular embodiments, the regulatorysequence can be a tissue specific promoter such that the expression ofthe protein will be substantially greater in the target tissue type(i.e., solid tumor) compared to other types of tissue. In particularembodiments, the regulatory sequence can result in the constitutiveexpression of the protein upon entry of the vector into the cell.Alternatively, the regulatory sequences can include inducible sequences.Inducible regulatory sequences are well known to those skilled in theart and are those sequences that require the presence of an additionalinducing factor to result in expression of protein. Examples of suitableregulatory sequences include binding sites corresponding totissue-specific transcription factors based on endogenous nuclearproteins, sequences that direct expression in a specific cell type, thelac operator, the tetracycline operator and the steroid hormoneoperator. Any inducible regulatory sequence known to those of skill inthe art may be used in conjunction with uses of compositions and methodsdisclosed herein.

In particular embodiments, the nucleic acid is stably integrated intothe genome of a subset of a subject's cells. In particular embodiments,the nucleic acid is stably maintained in a subset of a subject's cellsas a separate, episomal segment.

In particular embodiments, the efficiency of integration, the size ofthe nucleic acid sequence that can be integrated, and the number ofcopies of a nucleic acid sequence that can be integrated into a genomecan be improved by using transposons. Transposons or transposableelements include a short nucleic acid sequence with terminal repeatsequences upstream and downstream. Active transposons can encode enzymesthat facilitate the excision and insertion of nucleic acid into a targetnucleic acid (e.g., DNA) sequence.

A number of transposable elements have been described in the art thatfacilitate insertion of nucleic acids into the genome of vertebrates,including humans. Examples include sleeping beauty (e.g., derived fromthe genome of salmonid fish); piggyback (e.g., derived from lepidopterancells and/or the Myotis lucifugus); mariner (e.g., derived fromDrosophila); frog prince (e.g., derived from Rana pipiens); Tol2 (e.g.,derived from medaka fish); TcBuster (e.g., derived from the red flourbeetle Tribolium castaneum) and spinON.

In particular embodiments, active agent(s) are provided as conjugatesincluding an active agent(s) that targets CD11b/CD18, MPO, and/orIntegrin αVβ1 and a chemotherapeutic and/or cytotoxic agent. Examples ofchemotherapeutic/cytotoxic agents include capecitabine, paclitaxel,gemcitabine, methotrexate, doxorubicin, docetaxel, cisplatin, anddaunorubicin.

Regarding NIF particularly, and due to its size, additionalconsiderations may be warranted in formulation. Here, particularly,although useful with other active agent(s) as well, carrier moleculesfor effective delivery and hypersensitivity reduction can be used.Exemplary carrier molecules include liposomes, lipid micelles,lipoprotein micelles and polymeric micelles. Other carrier moleculesinclude polymer nanoparticles such as polycyanoacrylate nanoparticlesand PEGylated nanoparticles, polymer microparticles, block copolymermicelles, lipid stabilized emulsions, polymer-lipid hybrid systems andderivatized single chain polymers and other linear, spherical or tubularpolymers. The carriers may further include carbon or other nanotubesincluding linear and spherical, uni- and multiwalled nanotubes. Thecarriers may also include dendrimers such as PAMAM dendrimers, andfullerenes.

In particular embodiments incorporating proteins such as Abciximab, theproteins can be produced by continuous perfusion in mammalian cellculture. For Abciximab particularly, the 47,615 Dalton Fab fragment canbe purified. All relevant protein products can be purified from cellculture supernatant by a series of steps involving specific viralinactivation and removal procedures, digestion with papain and columnchromatography.

The present disclosure further provides for kits including one or moretreatment options (e.g., single or combination therapies) and/orchemoresistance detection assays for practicing any of the methodsdisclosed herein. The kits may include a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals, biological products, lab developed tests, etc., whichnotice reflects approval by the agency of the manufacture, use or salefor human administration and/or testing. Treatment portions of the kitsmay include agent(s) in a ready-to-use form and/or a form that requirespreparation before administration (e.g., lyophilized). Chemoresistancedetection assay portions of the kits may utilize any necessary orappropriate cellular assays, polypeptides, conjugates, antibodies,polynucleotides, expression vectors, cells, methods, compositions,systems, and/or apparatuses useful for the detection of chemoresistanceand/or C11b, CD18, CD51 and/or CD29.

The Exemplary Embodiments and Example below are included to demonstrateparticular embodiments of the disclosure. Those of ordinary skill in theart should recognize in light of the present disclosure that manychanges can be made to the specific embodiments disclosed herein andstill obtain a like or similar result without departing from the spiritand scope of the disclosure. More particularly, to the extent notpreviously stated herein, any molecule, agent, protein, peptide, orantibody that is capable of binding aberrantly expressed β2 integrin(CD11b/CD18), MPO, and/or Integrin αVβ1 in solid tumors or cells canserve as a therapeutic intervention providing one or more of prevention,treatment, vaccination, elimination or reduction of cancer cells,elimination or reduction of chemoresistant cells, elimination orreduction of cancer stem cells and/or elimination or reduction of benigntumors (e.g. fibroids).

The following description of terms supports the current disclosure.

Chemoresistant: The resistance of a cell to the actions of a chemicalcompound.

HOSEpiC: Human Ovarian Surface Epithelial Cells (HOSEpiC) provided byScienCell Research Laboratories from human ovarian tissue. HOSEpiC arecryopreserved at primary culture and delivered frozen. HOSEpiC areguaranteed to further expand for 5 population doublings in theconditions provided by ScienCell Research Laboratories. Used as controlcells.

MDAH-2774: MDAH-2774 (CRL-10303) cell line was obtained from AmericanType Culture Collection (ATCC, Manassas, Va.) and was developed fromcells in the ascitic fluid from a patient with endometrioid ovariancancer and forms tumors in nude mice.

SKOV-3: The SKOV-3 (HTB-75) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). A human adenocarcinoma cellline that was isolated from ascitic fluid of a 64 year old patient in1973. Grows in nude mice; forms moderately well differentiatedadenocarcinoma consistent with ovarian primary cancer. SKOV-3 cells areresistant to tumor necrosis factor and to several cytotoxic drugsincluding diphtheria toxin, cis-platinum and Adriamycin.

A2780: The A2780 human ovarian cancer cell line was established fromtumor tissue from an untreated patient and was obtained from SigmaAldrich. Cells grow as a monolayer. A2780 is the parent line to thecisplatin resistant cell line A2780 cis, which is resistant to 1 uMcisplatin.

OV-90: The OV-90 (CRL-11732) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). A human serous adenocarcinomacell line that was isolated from ascitic fluid of a 64 year old patientin 1992. The cells are tumorigenic in nude mice.

OV-21: The OV-21 (TOV-21G, CRL-11730) cell line was obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.). A humanadenocarcinoma cell line that was isolated from ascitic fluid of a 62year old patient in 1991. The cells are tumorigenic in nude mice.

OVCAR-3: The OVCAR-3 (HTB-161) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). A human adenocarcinoma cellline that was isolated from ascitic fluid of a 60 year old patient in1982. The cells are tumorigenic in nude mice. Cells are resistant toclinically relevant concentrations of adriamycin, melphalan andcisplatin.

T24: The T24 (HTB-4) cell line was obtained from American Type CultureCollection (ATCC, Manassas, Va.). The human urinary bladder transitionalcell carcinoma was derived from an 81 year old Caucasian female.

Caco-2: The Caco-2 (HTB-37) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). The colorectal adenocarcinomacell line was derived from a 72 year old Caucasian male.

RL95-2: The RL95-2 (CRL-1671) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). The endometrial carcinoma cellline was derived from a 65 year old Caucasian female.

NCI-H1688: The NCI-H1688 (CCL-257) cell line was obtained from AmericanType Culture Collection (ATCC, Manassas, Va.). The lung carcinoma cellline was derived from the metastatic site (liver) and is a classic smallcell lung cancer cell line. The cell line was obtained from a 50 yearold Caucasian male.

Hep G2: The HEP-G2 (HB-8065) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). The liver (hepatocellular)carcinoma cell line was derived from a 15 year old Caucasian male.

LNCaP clone FGC: The LNCaP clone FGC (CRL-1740) cell line was obtainedfrom American Type Culture Collection (ATCC, Manassas, Va.). Theprostate carcinoma cell line was derived from the metastatic site (leftsupraclavicular lymph node) of a 50 year old Caucasian male.

BxPC-3: The BxPC-3 (CRL-1687) cell line was obtained from American TypeCulture Collection (ATCC, Manassas, Va.). The pancreatic adenocarcinomacell line was derived from a 61 year old female.

Hydrogen Peroxide (H₂O₂): H₂O₂ is released following tissue injury andmay act as one of the early signals for the recruitment and activationof immune cells. The elevation of H₂O₂ in the cytosol of immune cellsappears to be a key signal that links a broad variety of biotic andabiotic stresses to the triggering of the inflammatory response.

Hypochlorous Acid (HOCl): Hypochlorous acid is a weak acid with thechemical formula HOCl. It forms when chlorine dissolves in water, and itis HOCl that actually does the disinfection when chlorine is used todisinfect water for human use.

Natural Killer Cell (NK Cells): NK cells are a type of cytotoxiclymphocyte critical to the innate immune system. The role NK cells playis analogous to that of cytotoxic T cells in the vertebrate adaptiveimmune response. NK cells provide rapid responses to viral-infectedcells and respond to tumor formation, acting at around 3 days afterinfection. NK cells are known to differentiate and mature in the bonemarrow, lymph nodes, spleen, tonsils, and thymus, where they then enterinto the circulation.

Polymorphonuclear Leukocyte (PMN): In common parlance, the termpolymorphonuclear leukocyte often refers specifically to neutrophilgranulocytes, the most abundant of the granulocytes; the other types(eosinophils, basophils, and mast cells) have lower numbers.Granulocytes are produced via granulopoiesis in the bone marrow.

Scratch Assay: In a scratch wound healing assay, a “wound gap” in a cellmonolayer is created by scratching utilizing a pipette tip, and the“healing” of this gap, which is 1 mm wide, by cell migration and growthtowards the center of the gap is monitored and quantitated.

Toll Like Receptor 4 (TLR-4): TLR 4 is a toll-like receptor. It detectslipopolysaccharide from Gram-negative bacteria and is thus important inthe activation of the innate immune system. Overexpression of TLR4 inhuman tumors often correlates with chemoresistance and metastasis.

Receptor Tyrosine Kinase Inhibitors (RTKI's): The protein kinaseinhibitors are a large group of unique and potent antineoplastic agentsthat specifically target protein kinases that are altered in cancercells and that account for some of their abnormal growth. Proteinkinases are ubiquitous intracellular and cell surface proteins that playcritical roles in cell signaling pathways involved in metabolism, injuryresponses, adaption, growth and differentiation. They act by adding aphosphate group to a protein (phosphorylation), usually on a specificamino acid, which often makes the protein or enzyme “active”. The humangenome has more than 500 protein kinases and they can be classified as(1) tyrosine, (2) serine-threonine or (3) nonspecific (both), based upontheir amino acid specificity.

EXEMPLARY EMBODIMENTS

1. A method of treating a solid tumor in a subject in need thereofincluding administering a therapeutically effective amount of one ormore active agent(s) that inhibits, suppresses and/or eliminatesCD11b/CD18, MPO, and/or Integrin αVβ1 activity in the subject, therebytreating the solid tumor.2. A method of embodiment 1 wherein the solid tumor is a benign solidtumor or a cancerous solid tumor.3. A method of embodiment 1 or 2 wherein the solid tumor is a benignfibroid tissue solid tumor.4. A method of embodiment 3 wherein the benign fibroid tissue solidtumor is a uterine fibroid.5. A method of any of embodiments 1-3 wherein the solid tumor causesadhesive capsulitis, arterial fibrosis, arthrofibrosis, Crohn's disease,cirrhosis, cystic fibrosis, endomyocardial fibrosis, fibrous cysts,idiopathic pulmonary fibrosis, keloids, mediastinal fibrosis,myelofibrosis, nephrogenic systemic fibrosis, Peyronie's disease,pulmonary fibrosis, progressive massive fibrosis, retroperitonealfibrosis, sclerodermalsystemic sclerosis, uterine fibroids (uterineleiomyomas), and/or a precancerous fibroma in the subject.6. A method of embodiment 2 wherein the cancerous solid tumor is apre-malignant solid tumor or a malignant solid tumor.7. A method of embodiment 6 wherein the cancerous solid tumor is achemoresistant solid tumor.8. A method of embodiment 6 or 7 wherein the cancerous solid tumor isacoustic neuroma, adenocarcinoma, astrocytoma, basal cell cancer, bileduct cancer, bladder cancer, breast cancer, bronchogenic cancer, centralnervous system cancer, cervical cancer, chondrosarcoma, choriocarcinoma,colon cancer, craniopharyngioma, ependymoma, Ewing's tumor,fibrosarcoma, glioma, hemangioblastoma, hepatocellular carcinoma,hepatoma, leiomyosarcoma, liposarcoma, lung cancer, melanoma,medulloblastoma, medullary cancer, medullary thyroid cancer, menangioma,mesothelioma, myxosarcoma, neuroblastoma, oligodendroglioma, osteogenicsarcoma, ovarian cancer, papillary adenocarcinomas, papillary thyroidcancer, pancreatic cancer, pheochromocytomas papillary cancer, pinealcancer, prostate cancer, renal cell cancer, retinoblastoma,rhabdomyosarcoma, sarcoma, sebaceous gland cancer, seminoma, squamouscell cancer, sweat gland cancer, synovioma, testicular tumor, testicularcancer, and/or Wilms' tumor.9. A method of any of embodiments 6-8 wherein the cancerous solid tumoris ovarian cancer or breast cancer.10. A method of any of embodiments 1-9 wherein the treating causes ananti-cancer effect in the subject.11. A method of any of embodiments 1-10 wherein the one or more activeagent(s) include a peptide, antibody, a small molecule, a fusionprotein, a conjugate, or a combination thereof.12. A method of any of embodiments 1-11 wherein the one or more activeagent(s) are selected from anti-CD11b antibodies, anti-MPO antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximab,neutrophil inhibitory factor (NIF) protein, human cathelicidin peptideLL-37, Clopidogrel, Cilostazol, Intetumumab, Abituzumab, or a compoundof formula (I)

wherein:B is absent and R¹ is phenyl; orB is methylene and R¹ is phenyl or phenyl substituted with one fluoro;N is nitrogen;X is selected from the group including O and S; andR³ is selected from the group including 4-carboxyphenyl and3-carboxy-4-chlorophenyl.13. A method of embodiment 12, wherein the compound is

14. A method of any of embodiments 1-12 wherein the one or more activeagent(s) are anti-CD11b antibodies, anti-MPO antibodies, anti-integrinαV antibodies, anti-integrin β1 antibodies, Abciximab and/or NIF.15. A method of any of embodiments 1-12 wherein said active agent isadministered in combination with a chemotherapeutic agent.16. A method of embodiment 15 wherein said chemotherapeutic agent iscisplatin.17. A method of embodiment 15 wherein said chemotherapeutic agent isdocetaxel.18. A method of any of embodiments 1-17 wherein said active agentsinclude anti-CD11b antibodies in combination with anti-MPO antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.19. A method of any of embodiments 1-18 wherein said active agentsinclude anti-MPO antibodies in combination with anti-CD11b antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.20. A method of any of embodiments 1-19 wherein said active agentsinclude anti-integrin αV antibodies in combination with anti-CD11bantibodies, anti-MPO antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.21. A method of any of embodiments 1-20 wherein said active agentsinclude anti-integrin β1 antibodies in combination with anti-CD11bantibodies, anti-MPO antibodies, anti-integrin αV antibodies, Abciximaband/or NIF.22. A method of any of embodiments 1-21 wherein said active agentsinclude Abciximab in combination with anti-CD11b antibodies, anti-MPOantibodies, anti-integrin αV antibodies, anti-integrin β1 antibodiesand/or NIF.23. A method of any of embodiments 1-22 wherein said active agentsinclude NIF in combination with anti-CD11b antibodies, anti-MPOantibodies, anti-integrin αV antibodies, anti-integrin β1 antibodiesand/or Abciximab.24. A method of any of embodiments 1-23 wherein the active agent(s) areadministered in protein or nucleic acid form.25. A method of any of embodiments 1-24 wherein the active agent(s) areprovided in therapeutically effective amounts as part of a composition.26. A composition including a therapeutically effective amount of one ormore active agent(s) that target CD11b/CD18, MPO, and/or Integrin αVβ1.27. A composition of embodiment 26 wherein the one or more activeagent(s) include a peptide, antibody, a small molecule, a fusionprotein, a conjugate, or a combination thereof.28. A composition of embodiment 26 or 27 wherein the one or more activeagent(s) are selected from anti-CD11b antibodies, anti-MPO antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximab,neutrophil inhibitory factor (NIF) protein, human cathelicidin peptideLL-37, Clopidogrel, Cilostazol, Intetumumab, Abituzumab, or a compoundof formula (I)

wherein:B is absent and R¹ is phenyl; orB is methylene and R¹ is phenyl or phenyl substituted with one fluoro;N is nitrogen;X is selected from the group including O and S; andR³ is selected from the group including 4-carboxyphenyl and3-carboxy-4-chlorophenyl.29. A composition of embodiment=28, wherein the compound is

30. A composition of any of embodiments 26-29 wherein the one or moreactive agent(s) include anti-CD11b antibodies, anti-MPO antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.31. A composition of any of embodiments 26-30 wherein said active agentsinclude anti-CD11b antibodies in combination with anti-MPO antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.32. A composition of any of embodiments 26-31 wherein said active agentsinclude anti-MPO antibodies in combination with anti-CD11b antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.33. A composition of any of embodiments 26-32 wherein said active agentsinclude anti-integrin αV antibodies in combination with anti-CD11bantibodies, anti-MPO antibodies, anti-integrin β1 antibodies, Abciximaband/or NIF.34. A composition of any of embodiments 26-33 wherein said active agentsinclude anti-integrin β1 antibodies in combination with anti-CD11bantibodies, anti-MPO antibodies, anti-integrin αV antibodies, Abciximaband/or NIF.35. A composition of any of embodiments 26-34 wherein said active agentsinclude Abciximab in combination with anti-CD11b antibodies, anti-MPOantibodies, anti-integrin αV antibodies, anti-integrin β1 antibodiesand/or NIF.36. A composition of any of embodiments 26-35 wherein said active agentsinclude NIF in combination with anti-CD11b antibodies, anti-MPOantibodies, anti-integrin αV antibodies, anti-integrin β1 antibodiesand/or Abciximab.37. A composition of any of embodiments 26-36 wherein thetherapeutically effective amount provides an anti-solid tumor effectand/or an anti-cancer effect when administered to a subject in needthereof.38. A method of killing chemoresistant cells in a subject in needthereof including administering a therapeutically effective amount ofone or more active agent(s) that target CD11b/CD18, MPO, and/or IntegrinαVβ1 to the subject, thereby killing the chemoresistant cells.39. A method of killing cancer stem cells in a subject in need thereofincluding administering a therapeutically effective amount of one ormore active agent(s) that target CD11b/CD18, MPO, and/or Integrin αVβ1lto the subject, thereby killing the cancer stem cells.40. A method of embodiment 38 or 39 wherein the one or more activeagent(s) include a peptide, antibody, a small molecule, a fusionprotein, a conjugate, or a combination thereof.41. A method of any of embodiments 38-40 wherein the one or more activeagent(s) are selected from anti-CD11b antibodies, anti-MPO antibodies,anti-integrin αV antibodies, anti-integrin β1 antibodies, Abciximab,neutrophil inhibitory factor (NIF) protein, human cathelicidin peptideLL-37, Clopidogrel, Cilostazol, Intetumumab, Abituzumab, or a compoundof formula (I)

wherein:B is absent and R¹ is phenyl; orB is methylene and R¹ is phenyl or phenyl substituted with one fluoro;N is nitrogen;X is selected from the group including O and S; andR³ is selected from the group including 4-carboxyphenyl and3-carboxy-4-chlorophenyl.42. A method of embodiment 41, wherein the compound is

43. A method of any of embodiments 38-42 wherein the one or more activeagent(s) are anti-CD11b antibodies, anti-MPO antibodies, anti-integrinαV antibodies, anti-integrin β1 antibodies, Abciximab and/or NIF.44. A method of any of embodiments 38-41 and 43 wherein the activeagent(s) are administered in protein or nucleic acid form.45. A method of any of embodiments 38-44 wherein the active agent(s) areprovided in therapeutically effective amounts as part of a composition.46. Use of Abciximab or neutrophil inhibitory factor (NIF) protein totreat ovarian cancer.

Example 1. Abciximab's Effects on Solid Tumors: Ovarian Cancer

A2780:

The A2780 human ovarian cancer cell line was established from tumortissue from an untreated patient and was obtained from Sigma Aldrich.Cells grow as a monolayer. A2780 is the parent line to the cisplatinresistant cell line A2780 cis, which is resistant to 1 μM cisplatin.This cell line is maintained in RPMI media (HyClone, Fisher Scientific)supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin and 10%heat-inactivated fetal bovine serum (FBS, Thermofisher Scientific) at37° C. in 5% CO₂. Culture medium was replaced every two days.

Cancer Stem Cells:

This cell line was derived from the commercially available SKOV-3 EOCcell line (ATCC). These cells were isolated from culture utilizing CD44and CD117 antibody-conjugated-magnetic beads and were found to expresspluripotency makers Oct4, Sox2, and NANOG. Cells are maintained inlow-bind 24 well culture dishes in DMEM/F12 media (Hyclone, Fisher)supplemented with 5 μg/ml Insulin, 20 ng/ml EGF, 10 ng/ml FGF, 0.5% BSA,and 1% penicillin and streptomycin at 37° C. in 5% CO₂. Healthy cellsform spheroids in culture, in low bind culture dishes.

Macrophages:

The commercial EL 1 (macrophage, ATCC) was established fromcryopreserved human spleen cells. Cells are maintained in Iscove'smodified Dulbecco's medium (ATCC) supplemented with 100 U/mL penicillinand 100 μg/mL streptomycin and 10% heat-inactivated fetal bovine serum(FBS, Thermofisher Scientific) at 37° C. in 5% CO₂. Culture medium wasreplaced every two days.

Cell Viability Assay:

Cell viability was determined using the TACS MTT Cell ProliferationAssay (Trevigen, Gaithersburg, Md.) per the manufacturer's protocol.Briefly, measurement of cell proliferation is based upon the reductionof the tetrazolium salt,3,[4,5-dimethytthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT). MTTis reduced to an insoluble formazan dye by mitochondrial enzymesassociated with metabolic activity of living cells. Cytotoxic compoundssuch as chemotherapeutics are able to damage and destroy cells, and thusdecrease the reduction of MTT to formazan. A standard curve with sevenpoints was constructed using each respective cell line. Cells wereseeded into 96-well plates in a fixed volume of 100 μl at a density of8000 cells per well, in triplicate. Cells were treated with eitherneutrophil inhibitory factor (10 μg) or increasing doses of Abciximabfor 24 or 72 hours in a cell culture incubator (37° C., 5% CO₂) with orwith chemotherapeutics. Following incubation, 10 μl of the MTT solutionwas added to each well and incubated for 2 hours followed by theaddition of 100 μl of the Detergent Solution to each well and incubationfor an additional 2 hours. Absorbance was measured at 570 nm. A blankcontaining only medium was subtracted from all test samples. A standardcurve was constructed utilizing the OD readings correlating with eachstandard point and was utilized to determine cell number in the testsamples.

In this Example, Abciximab was tested in normal human macrophages andHOSEpiC cells, sensitive EOC cell lines (A2780, MDAH-2774, SKOV-3, OV90,TOV21G, OV112D, OV433, and HTB-161) and their Taxotere or Cisplatinresistant counterparts, and other cancer cell lines including colon(HTB-37), endometrial (CRL-1671), lung (CCL-257), prostate (CRL-1740),bladder (HTB-4), and hepatocellular (HB-8065) cancers, in vitro. Theanti-cancer activity was significant in as compared to non-cancer (FIG.1A). There was a synergistic effect when Abciximab was combined withcisplatin in sensitive A2780 cells (FIGS. 1B-1D).

Example 2

Cell Viability was Determined Using the TACS MTT Cell ProliferationAssay (Trevigen, Gaithersburg, Md.) per the manufacturer's protocol.Briefly, measurement of cell proliferation is based upon the reductionof the tetrazolium salt,3,[4,5-dimethytthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT). MTTis reduced to an insoluble formazan dye by mitochondrial enzymesassociated with metabolic activity of living cells. A standard curve wasconstructed using each respective cell line using a two-fold dilutionseries. Cells were seeded into 96-well plates in a fixed volume of 100μl at a density of 8000 cells/well. Cells were treated with integrin αVor integrin β1, alone or in combination (0, 15, and 40 μg/ml) for 24hours. Following incubation, 10 μl of the MTT solution was added to eachwell and incubated for 2 hours followed by the addition of 100 μl of theDetergent Solution to each well and incubation for an additional 2-4hours. Absorbance was measured at 570 nm. A blank containing only mediumwas subtracted from all test samples.

RNA Isolation and Reverse Transcription of cDNA, for Real-Time RT-PCR.

RNA isolation. Total RNA was extracted from cells using the RNeasy MiniKit (Qiagen, Valencia, Calif.) according to the manufacturer's protocoland as previously reported in Eldh et al., Mol. Immunol., 2012. 50(4):p. 278-86.

Reverse Transcription and Real-Time RT-PCR.

Reverse transcription: A 20 μL cDNA reaction volume containing 1 μg RNAwas prepared using the SuperScript VILO Master Mix Kit (LifeTechnologies, Grand Island, N.Y.), as described by the manufacturer'sprotocol.

Real-Time RT-PCR Primer Design and Controls:

Optimal oligonucleotide primer pairs for real-time RT-PCR amplificationof reverse-transcribed cDNA were selected with the aid of the softwareprogram, Beacon Designer (Premier Biosoft Int., Palo Alto, Calif.).Human oligonucleotide primers, which amplify variable portions of theprotein coding regions, were used. Sequences of the oligonucleotidesused for amplification of β-actin, CD1 b, MPO, integrin αV and integrinβ1 mRNA are as described in Table 1 of FIG. 23.

Quantitative RT-PCR was performed using the EXPRESS SYBR GreenER qPCRSupermix Kit (Life Technologies) and the Cepheid 1.2f Detection System.Real-time RT-PCR was performed in a 25 μl total reaction volumeincluding 12.5 μl of EXPRESS SYBR GreenER qPCR Supermix, 1 μl of cDNAtemplate, and 0.2 μM each of target specific primers designed to amplifya part of each gene. Standards with known concentrations and lengths(base pairs (bp)) were designed specifically for each gene using theBeacon Designer software (Premier Biosoft, Palo Alto, Calif.), allowingfor construction of a standard curve using a tenfold dilution series. Aspecific standard for each gene allows for absolute quantification ofthe gene in number of copies, which can then be expressed per pg of RNA.Following real-time RT-PCR, a melting curve analysis was performed todemonstrate the specificity of the PCR product as a single peak. Sampleswere all normalized to A-actin. A control, containing all the reactioncomponents except for the template was included in all experiments. Allexperiments were performed in triplicate.

Results.

Cisplatin resistant A2780 cells have higher mRNA and protein levels ofintegrin αV and higher mRNA levels of integrin β1 as compared to theirchemosensitive counterparts. Treatment with antibodies integrin αV orintegrin β1 alone lead to a decrease in cell viability in a dosedependent manner. A combination of both antibodies lead to a furtherdecrease in viability in both sensitive and chemoresistant cell lines.

Example 3. MPO

MPO expression may be unique to EOC cells, as it was not detected inseveral other types of cancer cell lines (FIG. 5). These include theovarian cancer cell lines of SKOV-3 and MDAH-2774. Moreover, MPOexpression was not detectable in normal ovarian tissues (published).Immunoreactivity showed a striking increase in both CD11b and MPO inovarian cancer tissues as compared to normal ovarian tissues (FIG. 6).The presence of CD11b and MPO was also confirmed in EOC cells (FIG. 6).Additionally, further confirmation of the expression of both MPO andCD11b was determined in HOSEpiC as compared to EOC cells MDAH-2774 andSKOV-3 (FIGS. 7A and 7B). Additionally, CD11b is present in severaldifferent cancer cell tissues (FIG. 8). Utilizing CD44 and CD117antibody-conjugated magnetic beads, cancer stem-like cells (CSCs) wereisolated from the EOC cell line, SKOV-3. These CSCs were found toexpress both lower levels of CD11b and higher levels of MPO as comparedto SKOV-3 EOC cells. Without being bound by theory, it is believed thatwhen differentiated cancer cells die, they release cytokines and othercomponents that make the cancer stem like cells differentiate intomature cells, likely with aberrant genes and DNA/RNA/protein expressionand expression levels. CD11b antibody caused apoptosis in cells (FIGS.10A and 10B). This was determined by TUNEL assay, which detects damagedDNA and indicates apoptosis, as well as by an increase in caspase-3activity which correlates with increased apoptosis (FIGS. 10A and 10B).

Adding antibodies to cell cultures cause apoptosis in cells in the lowmicromolar range. On a scratch assay, the control cells grew back intothe scratch groove until contact inhibition was reestablished (FIG. 12).Those cells treated with the CD11b antibody did not grow back into thegroove. Further, the cells that were not part of the scratch started todie off, even at the low micromolar range (FIG. 12).

Animal Studies:

Growth of xenograph implants of SKOV-3 cells in athymic nude mice weresignificantly delayed when tumors were injected with CD11b antibody(FIG. 13). CD11b drastically decreases while MPO increases in all of thechemoresistant as compared to sensitive EOC cell lines tested(MDAH-2774, SKOV-3, A2780) (FIGS. 14A-14D). Additionally, CD18expression is lower in chemoresistant EOC cells as compared to sensitivecounterparts (FIG. 14E).

Viability Assays:

Treatment with CD11b antibody (15 μg/ml, 24 hours) resulted in no changein cell viability in either normal cells such as HOSEpiC and macrophages(FIG. 15A). Treatment of sensitive EOC cell lines (A2780, MDAH-2774,SKOV-3, OV90, TOV21G, OV112D, OV433, and HTB-161) and their Taxotere orCisplatin resistant counterparts with various doses of CD11b antibodyresulted in a significant reduction in cell viability (FIG. 15A). Othertypes of cancer cell lines including colon (HTB-37), endometrial(CRL-1671), lung (CCL-257), prostate (CRL-1740), bladder (HTB-4), andhepatocellular (HB-8065) cancers also had a significant reduction inviability upon treatment with increasing doses of the CD11b antibody.Combination of the CD11b antibody with cisplatin treatment of sensitiveand cisplatin resistant A2780 EOC cells resulted in a further increasein cytotoxicity and was observed to act in a synergistic fashion (FIGS.15B-15D). To test whether media collected from A2780 EOC cells containsa target that cross-reacts with the CD11b monoclonal antibody, cellswere cultured for 48 hours followed by removal of media. The CD11bantibody was incubated with the conditioned cell culture media for 24hours to block the antibody with the unknown target. The blockedantibody media was used to treat A2780 sensitive EOC cells for another24 hours followed by assessment of viability by the TACS MTT CellProliferation Assay. No decrease in cell viability was observed whentreating with media from cells containing “blocked” antibody (FIG. 16).

Treatment with the MPO antibody also decreased cell viability, albeit toa lesser extent than was observed with CD11b antibody treatment (FIGS.17A-17B). Combination of MPO antibody with increasing concentrations ofcisplatin was able to enhance cell death at the higher concentration ofMPO antibody (FIGS. 17C-17D).

CD11b has been implicated with TLR4 in ovarian cancer. As disclosedherein, expression of TLR4 increases in chemoresistant EOC cells ascompared to their chemosensitive counterparts (FIG. 18).

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.Thus, the terms “include” or “including” should be interpreted torecite: “comprise, consist of, or consist essentially of.” Thetransition term “comprise” or “comprises” means includes, but is notlimited to, and allows for the inclusion of unspecified elements, steps,ingredients, or components, even in major amounts. The transitionalphrase “consisting of” excludes any element, step, ingredient orcomponent not specified. The transition phrase “consisting essentiallyof” limits the scope of the embodiment to the specified elements, steps,ingredients or components and to those that do not materially affect theembodiment. In particular embodiments, lack of a material effect isevidenced by lack of a statistically-significant reduction in theembodiment's ability to kill ovarian cancer cells in vitro or in vivo.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents, printedpublications, journal articles and other written text throughout thisspecification (referenced materials herein). Each of the referencedmaterials are individually incorporated herein by reference in theirentirety for their referenced teaching.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the examples or when application of themeaning renders any construction meaningless or essentially meaningless.In cases where the construction of the term would render it meaninglessor essentially meaningless, the definition should be taken fromWebster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

ADDITIONAL REFERENCES

-   1. Rajput et al., “TLR is a Novel Determinant of the Response to    Paclitaxel in Breast Cancer,” Mol Cancer Ther., 12(8):1676-1687,    2013.-   2. Podolnikova et al., “Ligand Recognition Specificity of Leukocyte    Integrin αMβ2 (Mac-1, CD11b/CD18) and Its Functional Consequences,”    Biochemistry, 54(6):1408-1420, 2015.-   3. Sadhu et al, “CD11c/CD18: Novel Ligands and a Role in    Delayed-Type Hypersensitivity,” J Leuko Biol., 81(6):1395-1403,    2007.

What is claimed is:
 1. A method of inducing apoptosis of cells of anovarian tumor in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an anti-MPO antibodyand a therapeutically effective amount of an anti-CD11b antibody,thereby inducing apoptosis of cells of the ovarian tumor in the subject.2. The method of claim 1, further comprising administering atherapeutically effective amount of an anti-CD18 antibody.
 3. The methodof claim 1, further comprising administering a therapeutically effectiveamount of an anti-integrin aV antibody.
 4. The method of claim 1,further comprising administering a therapeutically effective amount ofan anti-integrin β1 antibody.
 5. The method of claim 1, furthercomprising administering a therapeutically effective amount of NIF. 6.The method of claim 1, further comprising administering atherapeutically effective amount of Abciximab.
 7. The method of claim 1,wherein the ovarian tumor is a pre-malignant solid tumor or a malignantsolid tumor.
 8. The method of claim 1, wherein the ovarian tumor is achemoresistant solid tumor.
 9. The method of claim 1, wherein theadministering causes an anti-cancer effect in the subject.
 10. Themethod of claim 1, wherein the anti-MPO antibody is administered as aprotein or as nucleic acid(s) encoding the protein.
 11. The method ofclaim 1, which is a method of treating ovarian cancer.
 12. The method ofclaim 11, wherein the ovarian cancer comprises chemosensitive cancercells.
 13. The method of claim 11, wherein the ovarian cancer compriseschemoresistant cancer cells.
 14. The method of claim 11, wherein theovarian cancer comprises cancer stem cells.
 15. The method of claim 2,wherein the anti-CD18 antibody is administered as a protein or asnucleic acid(s) encoding the protein.
 16. The method of claim 3, whereinthe anti-integrin aV antibody is administered as a protein or as nucleicacid(s) encoding the protein.
 17. The method of claim 4, wherein theanti-β1 antibody is administered as a protein or as nucleic acid(s)encoding the protein.
 18. The method of claim 5, wherein the NIF isadministered as a protein or as nucleic acid(s) encoding the protein.